Thio semicarbazone and semicarbozone inhibitors of cysteine proteases and methods of their use

ABSTRACT

The present invention relates to thio semicarbazone and semicarbazone inhibitors of cysteine proteases and methods of using such compounds to prevent and treat protozoan infections such as trypanosomiasis, malaria and leishmaniasis. The compounds also find use in inhibiting cysteine proteases associated with carcinogenesis, including cathepsins B and L.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. provisionalapplication Ser. No. 60/379,366 filed on May 8, 2002; and U.S.provisional application Ser. No. 60/449,058, filed Feb. 20, 2003; bothof which are herein incorporated by reference for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant Nos.1F-32-AI10293-02 and AI35707, awarded by the National Institutes ofHealth. The Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Trypanosomiasis, malaria, and leishmaniasis are major parasitic diseasesin developing countries (McKerrow, J. H. et al., Annu. Rev. Microbiol.47:821-853 (1993)). American trypanosomiasis, or Chagas' disease, is theleading cause of heart disease in Latin America (Libow, L. F. et al.,Cutis, 48:37-40 (1991)). At least 16-18 million people are infected withTrypanosoma cruzi, resulting in more than 50,000 deaths each year(Godal, T. et al., J. Tropical diseases. WHO Division of Control inTropical Diseases World Health Organization: Geneva, Switzerland, pp12-13. (1990); World Health Organization website:http://www.who.int/ctd/html/chagburtre.html). The statistics for malariaare more sobering, with about 300-500 million clinical cases and about 3million deaths each year. Further, at least 10 million people areinfected with a form of Leishmania each year (see Goodman & Gilman's ThePharmacological Basis of Therapeutics, 9^(th) Ed, 1996, McGraw-Hill, NewYork).

Chagas' disease is transmitted to humans by blood-sucking triatominevectors with an infectious trypomastigote form of the protozoan parasiteT. cruzi (Bonaldo, M. C. et al., Exp. Parasitol, 73:44-51 (1981); Harth,G., et al., T. Cruzi. Mol. Biochem Parasitol, 58:17-24 (1993);Meirelles, M. N. L., et al., Mol. Biochem. Parasitol, 52:175-184(1992)). African trypanosomiasis is transmitted to humans and cattle bytsetse flies and is caused by subspecies of T. brucei. So called“African sleeping sickness” is transmitted by an infectioustrypomastigote from T. brucei gambiense, and T. brucei rhodesienseproduces a progressive and usually fatal form of disease marked by earlyinvolvement of the central nervous system. T. brucei is further thecause of nagana in cattle, but bovine trypanosomiasis is alsotransmitted by T. congolense and T. evansi. In trypanosomiasisinfections, the trypomastigote enters the host bloodstream andultimately invades a cardiac muscle cell, where it transforms into theintracellular amastigote. The parasite may also be found in the blood,lymph, spinal fluid and cells of the gastrointestinal tract. Amastigotesreplicate within cells, transform back to trypomastigotes, and rupturethe cell, releasing the infectious form back into the bloodstream andother cells, amplifying the infection. Reviews of the currentunderstanding and treatment of African and American trypanosomiasisinfections is provided by Urbina (Curr Pharm Des (2002) 8:287) andBurchmore, et al (Curr Pharm Des (2002) 8:256).

Cruzain (aka cruzipain) is the major cysteine protease of T. cruzi. Theprotease is expressed in all life cycle stages of the parasite, butdelivered to different cellular compartments in each stage. In theepimastigote stage, which occurs in the insect vector, the protease isin a lysosomal compartment where it functions to degrade proteinsendocystosed from the insect gut. In the infectious trypomastigotestage, the protease appears at the flagellar pocket, the site ofendocytosis and secretion. In the amastigote stage, within the mammalianhost cell, the protease is both in the lysosomal compartment and on thesurface of the parasite where it may function in nutrition, remodelingof the mammalian cell, or evasion of host defense mechanisms. Additionof a cruzain inhibitor such as Z-Phe-Ala-FMK(benzyloxy-carbonyl-L-phenylalanyl L-alanine fluoromethyl ketone) tocultures of mammalian cells exposed to trypomastigotes or to mammaliancells already infected with T. cruzi amastigotes blocks replication anddifferentiation of the parasite (Bonaldo, M. C. et al., Exp. Parasitol,73:44-51 (1981); Harth, G., et al., T. Cruzi Mol. Biochem Parasitol,58:17-24 (1993); Meirelles, M. N. L., et al., Mol. Biochem. Parasitol,52:175-184 (1992)), thus arresting the parasite life cycle. Therefore,cruzain is essential for replication of the intracellular parasite.Treatment of T. cruzi-infected mice with a vinyl sulfone-derivatizedpseudopeptide inhibitor of cruzain, N-methylpiperazine-Phe-homoPhe-vinyl sulfone phenyl, has resulted in a cure in amouse model study (Engel, J. C. et al., J. Exp. Med., 188:725-734(1998)). Thus, cruzain is an appealing target for new antitrypanosomalchemotherapy (McKerrow, J. H. et al., Bioorg. Med. Chem., 7:639-644(1999)).

Malaria is caused by protozoa of the genus Plasmodium and is transmittedto humans through the bite of an infected anopheline mosquito. Theparasites develop into tissue schizonts in hepatic parenchymal cells,and then are released into the circulation as merozoites, which invadeerythrocytes. In erythrocytes, the merozoites mature from trophozoitesinto schizonts. Schizont-containing erythrocytes rupture to releasemerozoites that then invade more erythrocytes to continue the malarialcycle. Current understanding and treatment of plasmodium infections isreviewed in Winstanley (Lancet Infect Dis (2001) 1:206),Wongsrichanalai, et al (Lancet Infect Dis (2002) 2:209) and throughoutthe Feb. 7, 2002 issue of Nature (Lond) (vol. 415, issue 6872).

The majority of malaria infections is caused by Plasmodium falciparum(see Goodman & Gilman's The Pharmacological Basis of Therapeutics,supra). Papain-family cysteine proteases, known as falcipains, arehemoglobinases from P. falciparum that are essential to plasmodiumtrophozoite protein synthesis and development (Sijwali, et al (2001)Biochem J 360:481). Sequencing of the Plasmodium genome has revealed atleast three falcipain cysteine proteases, designated falcipain-1,falcipain-2 and falcipain-3, where falcipain-2 and falcipain-3 areunderstood to account for the majority of hemoglobinase activity in theplasmodium trophozoite (Joachimiak, et al (2001) Mol. Med 7:698). Thefalcipains are homologous to cruzain (Venturini, et al (2000) BiochemBiophys Res Commun 270:437 and Selzer, et al (1997) Exp Parasitol87:212) and at least the falcipain-2 sequence is highly conservedamongst different Plasmodium strains with different sensitivities toestablished antimalarial drugs (Singh and Rosenthal (2001) AntimicrobAgents Chemother 45:949). In in vitro studies, cysteine proteaseinhibitors blocked globin hydrolysis in Plasmodium infected erythrocytes(Rosenthal (1995) Exp. Parasitol 80:272 and Semenov et al (1998)Antimicrob Agents Chemother 42:2254). Importantly, oral or parenteraladministration of fluoromethyl ketone or vinyl sulfone peptidylinhibitors of falcipain cured treated mice that were infected withPlasmodium (Olson, et al (1999) Bioorg Med Chem 7:633). Therefore, thefalcipains and other homologous cysteine proteases are also importantantimalarial chemotherapeutic targets.

Leishmaniasis is caused by protozoal species and subspecies ofLeishmania transmitted to humans by the bites of infected femalephlebotamine sandflies. Promastigotes injected into the host arephagocytized by tissue monocytes and are transformed into amastigotes,which reside in intracellular phagolysosomes. Human leishmaniasis isclassified into cutaneous, mucocutaneous and visceral (kala azar) forms.Reviews of the current understanding and chemotherapy of leishmaniasisis provided by Croft and Yardley (Curr Pharm Des (2002) 8:319),Kafetzis, et al (Curr Opin Infect Dis (2002) 15:289, and Hepburn (CurrOpin Infect Dis 14:151).

In vitro and in vivo studies also have demonstrated that cysteineprotease inhibitors disrupt the infectious life cycle of Leishmania(see, Selzer, et al (1999) Proc Natl Acad Sci 96:11015; Das, et al(2001) J. Immunol 166:4020 and Salvati, et al (2001) Biochim BiophysActa 1545:357). Like Trypanosoma and Plasmodium, Leishmania synthesizecathepsin-L-like cysteine proteases that are essential to theirpathogenicity (Selzer, et al (1997) Exp Parasitol 87:212). The substraterecognition of one cysteine protease of L. mexicana, named CPB2.8 DeltaCTE, has been demonstrated to be similar to the substrate preferences ofcruzain (Alves, et al (2001) Mol Biochem Parasitol 117:137 and Alves, etal (2001) Mol Biochem Parasitol 116:1). Additionally, cruzain sharessequence similarity with homologous cysteine proteases from L. pifanoi,L. mexicana, and L. major (see Mottram, et al (1992) Mol Microbiol6:1925, Rafati, et al (2001) Mol Biochem Parasitol 113:35 and GenBanknumbers L29168, X62163 and AJ130942). Therefore, cysteine proteases alsorepresent a potential chemotherapeutic target against Leishmaniainfections.

Drugs currently used in the treatment of trypanosomiasis includeNifurtimox, Benznidazole, Suramin, Pentamidine isethionate, Eflornithineand Melarsoprol. Current chemotherapeutics for the treatment ofleishmaniasis include Stibogluconate sodium, Amphotericin B, andPentamidine isethionate. Drugs used in the treatment of malaria includechloroquine phosphate, mefloquine, halofantrine, and quinidine gluconatein combination with an antifolate or an antibiotic. Although theseprotozoans are inhibited to some extent by the administration ofavailable chemotherapeutics, the currently prescribed pharmacologicalcompounds to counteract trypanosomiasis, malaria, and leishmaniasis arelimited by the ability of the parasites to develop resistance to themand by their significant toxicity to the infected host. Therefore, thereis an interest in developing new drugs that will interfere with theinfectious life cycle of a parasite. Because cysteine proteases areessential to the life cycle of the parasites that cause trypanosomiasis,malaria and leishmaniasis, they are a logical target for newly developedchemotherapeutics (reviewed in Sajid and McKerrow, Mol Biochem Parasitol(2002) 120:1).

Several irreversible peptide-based inhibitor series including halomethylketones, diazomethanes, epoxysuccinyl derivatives, and vinyl sulfonederivatives targeting cysteine proteases have been developed (Otto, H.et al., Chem. Rev., 97:133-171 (1997)). A disadvantage of thechloromethyl ketones is their high reactivity and consequent lack ofselectivity. They react with serine proteases and other SH-containingmolecules, such as glutathione or nonproteolytic enzymes, and result intoxicity in vivo. To increase selectivity and reduce reactivity andtoxicity, a less reactive series of compounds, including monofluoromethyl ketones, epoxy derivatives (Roush, W. R. et al., Tetrahedron,56:9747-9762 (2000)), and vinyl sulfone derivatives (Bromme, D. et al.,Biochem. J., 315:85-89 (1996); Palmer, J. T. et al., J. Med Chem.38:3193-3196 (1995); Roush, W. R. et al., J. Am. Chem. Soc.120:10994-10995 (1998)) were developed. However, the low oralbioavailability associated with peptidyl inhibitors makes the furtherpursuit of effective chemical compounds of great interest.

Pharmaceutical compounds having a semicarbazone scaffold have beenevaluated for clinical use as an antihypertensive (Warren, J. D. et al.,J. Med. Chem., 20:1520-1521 (1977)), anticonvulsant (Dimmock, J. R. etal., Epilepsia, 35:648-655 (1994); Pandeya, S. N. et al., PharmacolRes., 37:17-22 (1998); Dimmock, J. R. et al., Drug Dev Res., 46:112-125(1999)), and antiallodynic agent (Carter, R. B. et al., Proceeding,International Symposium “Ion Channels in Pain and Neuroprotection” March14-17, San Francisco, Calif.; p 19 (1999)). For example, thesemicarbazone compound 4-[4-fluorophenoxy]benzaldehyde semicarbazone hasentered clinical trials for the treatment of neuropathic pain (Ramu, K.et al., Drug Metab. Dispos., 28:1153-1161 (2000)). Recently,5-nitrofurfural N-butyl semicarbazone (Cerecetto. H. et al., Farmaco,53:89-94 (1998); Cerecetto, H. et al., J. Med. Chem., 42:1941-1950(1999); Cerecetto, H. et al., Eur. J. Med. Chem., 35:343-350 (2000)) hasbeen shown to have antitrypanosomal activities targeting trypanothionereductase through a nitro anion radical mechanism, however, no cleartarget validation was reported in these papers.

Therefore, there is a pressing interest in developing potent,efficacious, economically synthesized pharmaceutical compounds withminimal toxicity and maximal bioavailability for the effective treatmentof these infectious parasitic diseases.

SUMMARY OF THE INVENTION

The present invention relates to thio semicarbazone and semicarbazonecompounds, and cyclized pyrazoline analogues of either, that function ascysteine protease inhibitors and the use of such compounds in methods oftreating and preventing protozoan infections that require cysteineprotease activity for their infectious lifecycle. The compounds alsofind use in inhibiting cysteine proteases associated with malignancy ofcancer cells.

In one aspect, the present invention relates to a method for inhibitinga cysteine protease involved in the infectious life cycle of a protozoanparasite, the method comprising the step of administering to the subjecta pharmaceutical composition comprising a pharmaceutically acceptablecarrier and a compound that inhibits such a cysteine protease, saidcomposition administered to the subject in an amount sufficient toinhibit the target cysteine protease and disrupt the infectious lifecycle of a protozoan parasite, wherein the compound forms a reversiblecovalent association with a cysteine in the active site of the targetcysteine protease.

In another aspect the present invention relates to a method for treatingor preventing a protozoan parasitic disease or infection, the methodcomprising the step of administering to the subject a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and acompound that inhibits a cysteine protease required in the infectiouslife cycle of the protozoan parasite, said composition administered tothe subject in an amount sufficient to inhibit the target cysteineprotease and disrupt the infectious life cycle of a protozoan parasitethereby treating or preventing a protozoan parasitic disease orinfection in the subject.

Exemplary protozoan cysteine proteases include those required in theinfectious life cycle of a trypanosome, such as cruzain or cruzipainfrom T. cruzi, rhodesain or brucipain from T. brucei rhodesiense, andcongopain from T. congolense; a plasmodium, such as falcipain from P.falciparum; or a leishmania, such as CPB2.8 Delta CTE from L. mexicana.

In one embodiment, the cysteine protease is a cathepsin L-like protease.

In one embodiment, the cysteine protease is a cathepsin B-like protease.

In one embodiment, the protozoan parasite is a Trypanosoma, a Plasmodiumor a Leishmania.

In a further embodiment, the parasite is selected from the groupconsisting of Trypanosoma cruzi, Trypanosoma brucei gambiense,Trypanosoma brucei rhodesiense, Trypanosoma rangeli, Trypanosomacongolense, Plasmodium falciparum, Plasmodium malariae, Plasmodiumvivax, Plasmodium ovale, Leishmania major, Leishmania braziliensis,Leishmania mexicana, Leishmania donvani, Leishmania pifanoi andLeishmania tropica.

In one embodiment the parasitic disease is selected from the groupconsisting of Chagas' disease, African sleeping sickness, nagana,malaria, and leishmaniasis (cutaneous, mucocutaneous or visceral).

In one aspect, the invention relates to a method of inhibiting amammalian cysteine protease involved in the malignancy of a cancer cell,the method comprising the step of administering to an individual in needthereof a composition comprising a pharmaceutically acceptable carrierand a compound that inhibits the mammalian cysteine protease by forminga reversible covalent association with a cysteine in the active site ofthe target cysteine protease.

In one embodiment, the cancer a breast cancer, an oral cancer, a skincancer, a lung cancer, an intestinal cancer, a bladder cancer or aprostate cancer. In a further embodiment, the cancer is a breastcarcinoma, an oral squamous cell carcinoma, a melanoma, a transitionalcell carcinoma of the bladder, an intestinal adenoma, a colorectalcarcinoma, a neuroblastoma or a prostate carcinoma. In anotherembodiment, the cancer is a metastatic cancer.

In another aspect, the invention relates to a method of inhibiting amammalian cysteine protease involved in an inflammatory disease, themethod comprising the step of administering to an individual in needthereof a composition comprising a pharmaceutically acceptable carrierand a compound that inhibits the mammalian cysteine protease by forminga reversible covalent association with a cysteine in the active site ofthe target cysteine protease.

In one embodiment the inflammatory disease is rheumatoid arthritis,atherosclerosis, vascular inflammation, allergic lung inflammation ormultiple organ failure.

In one embodiment, the mammalian cysteine protease is a cathepsin L, acathepsin B, a cathepsin H or a cathepsin K.

In one embodiment, the subject or individual is a mammal. In a furtherembodiment, the mammal is human, primate, canine, feline, equine,bovine, ovine, porcine, murine or lagomorpha.

The compounds are exemplified by a structural scaffold of the chemicalformula:

wherein

-   -   R¹ is a member selected from the group consisting of substituted        or unsubstituted aryl and substituted or unsubstituted        heteroaryl;    -   R² is substituted or unsubstituted alkyl, allyl, and alkyl        substituted with aryl;    -   R³ is a member selected from the group consisting of H, and        substituted or unsubstituted lower alkyl, and R² and R³ are        optionally joined to form a ring system having the formula:    -   R⁴ is a member selected from H, alkyl substituted with saturated        rings, aryl, or unsubstituted lower alkyl;    -   R⁵ is a member selected from the group consisting of H, aryl,        and substituted or unsubstituted alkyl; and X is O or S.        Preferably, the compounds inhibit a target cysteine protease of        interest with a potency or IC50 value of less than 1000        nanomolar (nM), more preferably of less than about 500, 300 or        100 nM, and most preferably less than about 80, 70, 60, 50, 40,        30, 20, or 10 nM.

In one embodiment, R¹ is a member selected from the group consisting of:

wherein

-   -   R⁵, R⁶, and R⁷ are members independently selected from the group        consisting of H, substituted or unsubstituted alkyl, substituted        or unsubstituted aryl, substituted or unsubstituted heteroaryl,        haloalkyl, alkoxy and halo; and Z is S or O.

In a further embodiment, R¹ is:

-   -   wherein R⁵, R⁶ and R⁷ are members independently selected from H,        halo and haloalkyl.

In a further embodiment, R⁵ is H or haloalkyl; R⁶ is H or halo; and R⁷is halo or CF₃.

In one embodiment, R² is a member selected from the group consisting ofH, CH₃, and CH₂CH₃.

In one embodiment, R⁵ is a member selected from the group consisting ofH, CH₃, and CH₂CH₃.

In one aspect, the compounds are provided as part of a pharmaceuticalcomposition. An exemplified pharmaceutical composition includes at leastone compound such as a thio semicarbazone, a semicarbazone or a cyclizedpyrazoline analogue of either, in a pharmaceutically acceptable carrier,such that administration of the composition to an individual wouldrender the one or more compounds sufficiently bioavailable toeffectively inhibit a target cysteine protease.

In one embodiment, the one or more compounds inhibit a target cysteineprotease without inducing toxicity in a host cell or host tissueinfected with a protozoan whose infectious life cycle requires theactivity of the target protease or in a non-malignant host cell or hosttissue that does not express a cysteine protease associated with amalignant cancer cell. Generally, the compounds form a reversiblecovalent interaction with a cysteine in the active site of a targetcysteine protease as part of their inhibitory mechanism.

In one embodiment, the compounds are at least one of Trypanocidal,Plasmodium-cidal, and Leishmania-cidal.

In one embodiment, the compounds are at least one of Trypanostatic,Plasmodium-static, and Leishmania-static.

Exemplary compounds, which find particular use in a pharmaceuticalcomposition of the invention, include:

-   a) 3′-Bromopropiophenone Thio Semicarbazone (1i),-   b) 3′-Chloropropiophenone Thio Semicarbazone (2a),-   c) 3′-Trifluoromethylpropiophenone Thio Semicarbazone (2b),-   d) 3′-Bromoacetophenone Thio Semicarbazone (3b),-   e) 3,4-Dichlorobenzaldehyde Thio Semicarbazone (3g)-   f) 3′,4′-Dichloroacetophenone Thio Semicarbazone (3h),-   g) 3-(3-Bromophenyl)-4-methyl-2-pyrazoline-1-thiocarboxamide (4b),-   h) 3-(3-Chlorophenyl)-4-methyl-2-pyrazoline-1-thiocarboxamide (4d),-   i) 3-(3,4-Dichlorophenyl)-2-pyrazoline-1-thiocarboxamide (4e),-   j)    3-(3-Trifluoromethylphenyl)-4-methyl-2-pyrazoline-1-thiocarboxamide    (4h),-   k) 3′,5′-bis(trifluoromethyl)propiophenone Thio Semicarbazone (2h),-   l) 3′,4′-Dichloropropiophenone Thio Semicarbazone (2i),-   m) 3-Trifluoromethylacetophenone Thio Semicarbazone (3d),-   n) 3′,5′-Bis(trifluoromethyl)acetophenone Thio Semicarbazone (3f),-   o) 3-(3,4-Dichlorophenyl)-4-methyl-2-pyrazoline-1-thiocarboxamide    (4f), and-   p) 3-(3-Trifluoromethylphenyl)-2-pyrazoline-1-thiocarboxamide (4g).

Compounds of further interest include:

-   4-(Phenylethynyl)thiophene-2-carboxyaldehyde Thio Semicarbazone    (1a),-   5-(4-Chlorophenyl)-2-furancarboxaldehyde Thio Semicarbazone (1b),-   5-(2-Methoxyphenyl)-2-furancarboxaldehyde Thio Semicarbazone (1c),-   2-(1-Methyl-3-trifluoromethyl)pyrazol-5-yl)-thiophene-5-carboxaldehyde    Thio Semicarbazone (1d),-   5-(3-Chlorophenyl)-2-furancarboxaldehyde Thio Semicarbazone (1e),-   5-(4-Bromophenyl)-2-furancarboxaldehyde Thio Semicarbazone (1f),-   1-(Phenylsulfonyl)-2-pyrrolecarboxaldehyde Thio Semicarbazone (1g),-   4′-Morpholinoacetophenone Thio Semicarbazone (1h),-   3-Hydroxy-4-methoxybenzaldehyde Thio Semicarbazone (1j),-   6′-Methoxy-2′-propiononaphthone Thio Semicarbazone (1k),-   4-Dimethylamino-1-naphthaldehyde Thio Semicarbazone (1l),-   1-Methylindole-3-carboxaldehyde Thio Semicarbazone (1m),-   2-Methoxy-1-naphthaldehyde Thio Semicarbazone (1n),-   3′-Fluoropropiophenone Thio Semicarbazone (2c),-   Propiophenone Thio Semicarbazone (2d),-   4′-Bromopropiophenone Thio Semicarbazone (2e),-   4′-Chloropropiophenone Thio Semicarbazone (2f),-   4′-Methoxypropiophenone Thio Semicarbazone (2g),-   2′,4′-Dichloropropiophenone Thio Semicarbazone (2j),-   3′-Methoxyacetophenone Thio Semicarbazone (2k),-   2′-Bromoacetophenone Thio Semicarbazone (2l),-   2′-Chloroacetophenone Thio Semicarbazone (2m),-   2,3-Dichlorobenzaldehyde Thio Semicarbazone (2n),-   3,5-Dichlorobenzaldehyde Thio Semicarbazone (2o),-   2-Trifluoromethylbenzaldehyde Thio Semicarbazone (2p),-   3-Bromobenzaldehyde Thio Semicarbazone (3a),-   3-Trifluoromethylbenzaldehyde Thio Semicarbazone (3c),-   3,5-Bis(trifluoromethyl)benzaldehyde Thio Semicarbazone (3e),-   2-Acetyl-5-bromothiophene Thio Semicarbazone (3i),-   2-Acetyl-5-chlorothiophene Thio Semicarbazone (3j),-   3-(3-Bromophenyl)-2-pyrazoline-1-thiocarboxamide (4a),-   3-(3-Chlorophenyl)-2-pyrazoline-1-thiocarboxamide (4c),-   3′-Bromopropiophenone N-Methyl Thio Semicarbazone (5a),-   3-(3-Bromophenyl)-2-pyrazoline-1-(N-methyl)thiocarboxamide (5b),-   3′-Bromopropiophenone N-(4-Trifluoromethylphenyl) Thio Semicarbazone    (5c),-   3-(3-Bromophenyl)-2-pyrazoline-1-(IL3-chlorophenyl)thiocarboxamide    (5d),-   3-(3-Chlorophenyl)-2-pyrazoline-1-(N-3-tri-fluoromethylphenyl)thiocarboxamide    (5e),-   3-(3-Bromophenyl)-2-pyrazoline-1-(N-hexyl)-thiocarboxamide (5f),-   3′-Bromopropiophenone Semicarbazone (6a),-   3′-Bromoacetophenone Semicarbazone (6b) and-   5-(3-Chlorophenyl)-2-furancarboxaldehyde Semicarbazone (6c)-   3′-Bromobutyrophenone thio Semicarbazone (8a)-   3′-Bromovalerophenone thio Semicarbazone (8b)-   1-(3′-Bromophenyl)-3-butenone thio Semicarbazone (8c)-   1-(3′-Bromophenyl)-2-phenylethanone thio Semicarbazone (8d)-   1-(3′-Bromophenyl)-3-phenylpropanone thio Semicarbazone (8e)-   3′-trifluoromethylbutyrophenone thio Semicarbazone (8f)-   3′-trifluoromethylvalerophenone thio Semicarbazone (8g)-   1-(3′-trifluoromethyl)-3-butenone thio Semicarbazone (8h)-   1-(3′-trifluoromethyl)-2-phenylethanone thio Semicarbazone (8i)-   1-(3′-trifluoromethyl)-3-phenylpropanone thio Semicarbazone (8j)-   3-(3-trifluoromethylphenyl)-4-ethyl-2-pyrazoline-1-Thiocarboxamide    (8k)-   3-(3-trifluoromethylphenyl)-4-propyl-2-pyrazoline-1-Thiocarboxamide    (8l)-   3-(3-trifluoromethylphenyl)-4-phenyl-2-pyrazoline-1-Thiocarboxamide    (8m)-   3-(3-bromophenyl)-4-benzyl-2-pyrazoline-1-Thiocarboxamide (8n)

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a thio semicarbazone scaffold.

FIG. 2 shows the initial 14 thio semicarbazone compounds synthesized andtheir inhibition of cruzain activity as represented by IC50 values.

FIG. 3 shows how variation of the aryl substituents and position of thesubstituents effects different cruzain inhibitory capacities. Compoundsin bold have lower (better) IC50 values than compound 1i.

FIG. 4 shows that compound having a C-5 ethyl group inhibit cruzain moreeffectively than compounds with a C-5 methyl group or a C-5 hydrogen.

FIG. 5 shows that cyclized pyrazoline analogues are generally potentinhibitors of cruzain. Compounds in bold have lower (better) IC50 valuesthan compound 1i.

FIG. 6 shows that substitution on the N1 amino group results inhibitorswith higher IC50 values in comparison to the IC50 value of compound 1i.

FIG. 7 shows that semicarbazone analogues of the most potent thiosemicarbazones poorly inhibit cruzain, as indicated by the high IC50values.

FIG. 8 shows the cell culture activity of representative thiosemicarbazones.

FIG. 9 shows the cell culture activity of additional thio semicarbazonecompounds with low IC50 values.

FIG. 10 shows a line graph depicting IC50 values versus time. The dataof representative thio semicarbazones are shown.

FIG. 11 shows a tridimensional depiction of the best calculatedorientation of compound 1i in the active site of cruzain. The surface ofthe active site of cruzain is represented by gray dots. His159 and Cys25are colored in green. Compound 1i is colored according to atom types:bromine, magenta; carbon, gray; hydrogen, white; nitrogen, blue; sulfur,yellow.

FIG. 12 shows a proposed mechanism of reversible covalent interactionbetween thio semicarbazones and cruzain based on experimental andmodeling results.

FIG. 13 shows the synthesis of compounds with additional C5-side chainvariations.

FIG. 14 shows the IC50s of the compounds with additional C5-side chainvariations. This is consistent with our modeling efforts that show theC5-side chain occupying the shallow S1 pocket, and therefore accepting awide range of groups without greatly decreasing the IC50s.

FIG. 15 shows in vitro data and IC50s for compounds.

DETAILED DESCRIPTION OF THE INVENTION

Introduction

Trypanosoma, Leishmania and Plasmodium infections require the action ofa parasitic cysteine protease to complete their infectious life cycle.In this application, we report that thio semicarbazones and relatedanalogues represent a novel series of small-molecule compounds thatdisrupt the parasitic infectious life cycle through inhibition ofessential cysteine proteases and serve as promising agents forantiparasitic therapy. These inhibitors of cysteine proteases, includingcathepsin-L like cysteine proteases, have specific structure activityrelationships which will allow for the development of additional potentinhibitors of cysteine proteases involved in parasitic disease statessuch as malaria, leishmaniasis and trypanosomiasis (e.g., Chagas'disease). The advantages of the compounds are many, including (i)minimal cellular toxicity, (ii) physical properties compatible withdesirable pharmacokinetics (low molecular weight, favorable C log P,favorable hydrogen bond donating and accepting capabilities), (iii) highpotency of target inhibition, with IC₅₀ values at the low nanomolarlevel, (iv) parasiticidal and parasitistatic efficacy against parasiteinfections of cells, (v) efficient synthesis and inexpensive production,and (vi) improved bioavailability over peptidyl inhibitors. Theparasiticidal activity of the thio semicarbazones represents asignificant advance. For example, the most potent and efficaciousacylhydrazide- and urea-based cathepsin-L like inhibitors are onlytrypanostatic for 18 and 22 days, respectively.

Thio semicarbazone compounds also find use in the inhibition of relatedmammalian cysteine proteases, including cathepsin L, cathepsin B,cathepsin H, cathepsin K and cathepsin S. The catalytic activities ofthese cysteine proteases have been implicated in the pathogenesis ofcancer and inflammation (reviewed in Banks, et al, Adv Exp Med Biol(2000) 477:349 and Dickenson Crit Rev Oral Biol Med (2002) 13:238). Forexample, cathepsin L, cathepsin B and cathepsin H are used ascorrelative prognostic indicators of the increased invasiveness,malignancy and growth status of numerous cancers including breastcarcinoma, oral squamous cell carcinoma, melanoma, transitional cellcarcinoma of the bladder, intestinal adenoma, colorectal carcinoma,neuroblastoma and prostate carcinoma (see, for example, Kawasaki, et al,Oral Surg Oral Med Oral Pathol Oral Radiol Endod (2002) 93:446; Staack,et al, Urology (2002) 59:308; Levicar, et al, Cancer Detect Prev (2002)26:42; Marten, et al, Gastroenterology (2002) 122:406; Jagoe, et al,Clin Sci (Lond) (2002) 102:353; Sinha, et al, Cancer (2002) 94:3141;Castino, et al, Int J Cancer (2002) 97:775; Waghray, et al, J Biol Chem(2002) 277:11533; Murnane, et al, Cancer Res (1991) 51:1137; Shuja, etal, Int J Cancer (1991) 49:341; Foekens, et al, J Clin Oncol (1998)16:1013; Friedrich, et al, Eur J Cancer (1999) 35:138; Kos, et al, ClinCancer Res (1997) 3:1815; and Strojan, et al, Clin Cancer Res (2000)6:1052). Additionally, mRNA and protein expression of cathepsin K, alikely contributor to bone metastasis in breast cancer, has beenobserved in breast cancer cells (Ishikawa, et al, Mol Carcinog (2001)32:84). With regard to inflammatory conditions, the catalytic action ofcathepsin B has been associated with rheumatoid arthritis,atherosclerotic plaque rupture and vascular inflammation, T cellmigration in allergic lung inflammation, and as a contributing proteaseto multiple organ failure (see, Zwicky, et al, Biochem J (2002)367:209-217; Chen, et al, Circulation (2002) 105:2766; Layton, et al,Inflamm Res (2001) 50:400; and Jochum, et al, Am J Respir Crit Care Med(1994) 150:S 123). Additionally, chronic inflammation and extracellularmatrix degradation preliminary to abdominal aortic aneurysm isassociated with a 30-fold increased transcriptional expression ofcathepsin H (Tung, et al, J Vasc Surg (2001) 34:143). Numerous studieshave demonstrated the efficacy of inhibiting cathepsin L or cathepsin Bin counteracting pathogenic processes of cancer and inflammation (see,for example, Katunuma, et al, Arch Biochem Biophys (2002) 397:305;Katunuma, et al, Adv Enzyme Regul (2002) 42:159; Greenspan, et al, J MedChem (2001) 44:4524; Kobayashi, et al, Cancer Res (1992) 52:3610;Kolkhorst, V, et al, J Cancer Res Clin Oncol (1998) 124:598; Krueger, etal, Cancer Res (1999) 59:6010; Sexton and Cox, Melanoma Res (1997) 7:97;Cox, et al, Melanoma Res (1999) 9:369; Castino, et al, supra; andLayton, et al, supra).

Definitions

The terms “cysteine protease” or “cysteine proteinase” or “cysteinepeptidase” intend any enzyme of the sub-subclass EC 3.4.22, whichconsists of proteinases characterized by having a cysteine residue atthe active site and by being irreversibly inhibited by sulfhydrylreagents such as iodoacetate. Mechanistically, in catalyzing thecleavage of a peptide amide bond, cysteine proteases form a covalentintermediate, called an acyl enzyme, that involves a cysteine and ahistidine residue in the active site (Cys25 and His159 according topapain numbering, for example). Cysteine protease targets of particularinterest in the present invention belong to the family C1 within thepapain-like clan CA. Representative cysteine protease targets for thepresent invention include papain, cathepsin B (EC 3.4.22.1), cathepsin H(EC 3.4.22.16), cathepsin L (EC 3.4.22.15), cathepsin K, cathepsin S (EC3.4.22.27), cruzain or cruzipain, rhodesain, brucipain, congopain,falcipain and CPB2.8 Delta CTE. Preferred cysteine protease targets ofthe present invention cleave substrate amino acid sequences-Phe-Arg-|-Xaa-, -Arg-Arg-|-Xaa-, -Val-Val-Arg-|-Xaa- or-Gly-Pro-Arg-|-Xaa-. Clan CA proteases are characterized by theirsensitivity to the general cysteine protease inhibitor, E64(L-trans-epoxysuccinyl-leucyl-amido (4-guanidino)butane) and by havingsubstrate specificity defined by the S₂ pocket.

Cysteine proteases of the present invention can be “cathepsin L-like” or“cathepsin B-like.” A cathepsin L-like cysteine protease sharesstructural and functional similarity with a mammalian cathepsin L, andcomprises a “ERFNIN” motif (Sajid and McKerrow, supra). Cathepsin L-likecysteine proteases prefer as a substrate the dipeptide sequence-Phe-Arg-|-Xaa-. Representative cathepsin L-like cysteine proteasesinclude cathepsin L, cathepsin K, cathepsin S, cruzain, rhodesain andcongopain, T. cruzi-L, T. rangeli-L, T. congolense-L, T. brucei-L, P.falciparum-L1, P. falciparum-L2, P. falciparum-L3, P. vivax-L1, P.cynomolgi-L1, P. vinckei-L and L. major-L. A cathepsin B-like cysteineprotease shares structural and functional similarity with a mammaliancathepsin B, and comprises an “occluding loop” (Sajid and McKerrow,supra). Cathepsin B-like cysteine proteases cleave as a substrate thedipeptide sequences -Arg-Arg-|-Xaa- and -Phe-Arg-|-Xaa-. Representativecathepsin B-like proteases include cathepsin B, T. cruzi-B, L.mexicana-B and L. major-B.

“Inhibitors” or “inhibition” of cysteine proteases refers to inhibitorycompounds identified using in vitro and in vivo assays for cysteineprotease function. In particular, inhibitors refer to compounds thatdecrease or obliterate the catalytic function of the target cysteineprotease, thereby interfering with or preventing the infectious lifecycle of a parasite or the migratory capacity of a cancer cell or aninflammatory cell. In vitro assays evaluate the capacity of a compoundto inhibit the ability of a target cysteine to catalyze the cleavage ofa test substrate. Cellular assays evaluate the ability of a compound tointerfere with the infectious life cycle of a parasite or the migrationof a cancer or inflammatory cell ex vivo, while not exhibiting toxicityagainst the host cell. Cellular assays measure the survival of aparasite-infected cell in culture. Preferred inhibitors allow forextended survival of an infected cell, either by delaying the life cycleof the parasite, or by killing the parasite. In vivo assays evaluate theefficacy of test compounds to prevent or ameliorate disease symptoms,such as those associated with parasitic infection, cancer invasion orgrowth, or inflammatory cell migration. Inhibitors are compounds thateliminate or diminish the catalytic function of a cysteine protease.Further, preferred inhibitors delay, interfere with, prevent oreliminate the completion of the infectious life cycle of a parasite orthe migratory ability of a cancer cell or an inflammation cell.Additionally, preferred inhibitors prevent or diminish a parasiticinfection in an individual or the migration of cancer cells orinflammatory cells in an individual, thereby preventing or amelioratingthe pathogenic symptoms associated with such infections or the migrationof rogue cells.

To examine the extent of inhibition, samples, assays, cultures or testsubjects comprising a target cysteine protease are treated with apotential inhibitor compound and are compared to negative controlsamples without the test compound, and positive control samples, treatedwith a compound known to inhibit the target cystein protease. Negativecontrol samples (not treated with a test compound), are assigned arelative cysteine protease activity level of 100%. Inhibition of acysteine protease is achieved when the cysteine protease activityrelative to the control is about 90%, preferably 75% or 50%, morepreferably 25-0%.

An amount of compound that inhibits a cysteine protease, as describedabove, is “an amount sufficient to inhibit a cysteine protease”, or a“cysteine protease inhibiting amount” of compound, thereby preventing ortreating a parasitic infection, inflammation, or cancer invasion orgrowth in an individual.

The term “IC50” refers to the concentration of compound that results inhalf-maximal inhibition of enzyme.

By “parasitistatic” or “trypanostatic” or “plasmodium-static” or“leishmania-static” is intended that the intracellular cycle of theparasite is completed at a slower growth rate and the infected hostcells survive longer.

The term “parasiticidal” or “trypanocidal” or “plasmodium-cidal” orleishmania-cidal” means that the intracellular cycle of the parasite isnot completed, therefore, leading to the death of the parasites.

For the thio semicarbazone compounds of the invention, the term “alkyl,”by itself or as part of another substituent, means, unless otherwisestated, a straight or branched chain, or cyclic hydrocarbon radical, orcombination thereof, which may be fully saturated, mono- orpolyunsaturated and can include di- and multivalent radicals, having thenumber of carbon atoms designated (i.e., C1-C10 means one to tencarbons). Examples of saturated hydrocarbon radicals include groups suchas methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologsand isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, andthe like. An unsaturated alkyl group is one having one or more doublebonds or triple bonds. Examples of unsaturated alkyl groups includevinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. The term “alkyl,” unlessotherwise noted, is also meant to include those derivatives of alkyldefined in more detail below as “heteroalkyl.” Alkyl groups which arelimited to hydrocarbon groups are termed “homoalkyl”.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkane, as exemplified by—CH2CH2CH2CH2-, and further includes those groups described below as“heteroalkylene.” Typically, an alkyl (or alkylene) group will have from1 to 24 carbon atoms, with those groups having 10 or fewer carbon atomsbeing preferred in the present invention. A “lower alkyl” or “loweralkylene” is a shorter chain alkyl or alkylene group, generally havingeight or fewer carbon atoms.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and from one to three heteroatoms selectedfrom the group consisting of O, N, Si and S, and wherein the nitrogenand sulfur atoms may optionally be oxidized and the nitrogen heteroatommay optionally be quaternized. The heteroatom(s) O, N and S may beplaced at any interior position of the heteroalkyl group. The heteroatomSi may be placed at any position of the heteroalkyl group, including theposition at which the alkyl group is attached to the remainder of themolecule. Examples include —CH2-CH2-O—CH3, —CH2-CH2-NH—CH3,—CH2-CH2-N(CH3)-CH3, —CH2-S—CH2-CH3, —CH2-CH2, —S(O)—CH3,—CH2-CH2-S(O)2-CH3, —CH═CH—O—CH3, —Si(CH3)3, —CH2-CH═N—OCH3, and—CH═CH—N(CH3)-CH3. Up to two heteroatoms may be consecutive, such as,for example, —CH2-NH—OCH3 and —CH2-O—Si(CH3)3. Similarly, the term“heteroalkylene” by itself or as part of another substituent means adivalent radical derived from heteroalkyl, as exemplified by—CH2-CH2-S—CH2CH2- and —CH2-S—CH2-CH2-NH—CH2-. For heteroalkylenegroups, heteroatoms can also occupy either or both of the chain termini(e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, andthe like). Still further, for alkylene and heteroalkylene linkinggroups, no orientation of the linking group is implied.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include cyclopentyl, cyclohexyl, 1-cyclohexenyl,3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkylinclude 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C1-C4)alkyl” is mean to include trifluoromethyl,2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,typically aromatic, hydrocarbon substituent which can be a single ringor multiple rings (up to three rings) which are fused together or linkedcovalently. The term “heteroaryl” refers to aryl groups (or rings) thatcontain from zero to four heteroatoms selected from N, O, and S, whereinthe nitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. A heteroaryl group can be attachedto the remainder of the molecule through a heteroatom. Non-limitingexamples of aryl and heteroaryl groups include phenyl, 1-naphthyl,2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of the above notedaryl and heteroaryl ring systems are selected from the group ofacceptable substituents described below.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) are meant to include both substituted and unsubstitutedforms of the indicated radical. Preferred substituents for each type ofradical are provided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be a variety of groups selected from: —OR′, ═O,═NR′, ═N—OR′, —NR′R″, —SR′9, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′,—CO2R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)2R′,—NH—C(NH2)═NH, —NR′C(NH2)═NH, —NH—C(NH2)═NR′, —S(O)R′, —S(O)2R′,—S(O)2NR′R″, —CN and —NO2 in a number ranging from zero to (2m′+1),where m′ is the total number of carbon atoms in such radical. R′, R″ andR′″ each independently refer to hydrogen, unsubstituted (C1-C8)alkyl andheteroalkyl, unsubstituted aryl, aryl substituted with 1-3 halogens,unsubstituted alkyl, alkoxy or thioalkoxy groups, or aryl-(C1-C4)alkylgroups. When R′ and R″ are attached to the same nitrogen atom, they canbe combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.For example, —NR′R″ is meant to include 1-pyrrolidinyl and4-morpholinyl. From the above discussion of substituents, one of skillin the art will understand that the term “alkyl” is meant to includegroups such as haloalkyl (e.g., —CF3 and —CH2CF3) and acyl (e.g.,—C(O)CH3, —C(O)CF3, —C(O)CH2OCH3, and the like).

Similarly, substituents for the aryl and heteroaryl groups are variedand are selected from: -halogen, —OR′, —OC(O)R′, —NR′R″, —SR′, —R′, —CN,—NO2, —CO2R′, —CONR′R″, —C(O)R′, —OC(O)NR′R″, —NR″C(O)R′, —NR″C(O)2R′,—NR′—C(O)NR″R′″, —NH—C(NH2)═NH, —NR′C(NH2)═NH, —NH—C(NH2)═NR′, —S(O)R′,—S(O)2R′, —S(O)2NR′R″, —N3, —CH(Ph)2, perfluoro(C1-C4)alkoxy, andperfluoro(C1-C4)alkyl, in a number ranging from zero to the total numberof open valences on the aromatic ring system; and where R′, R″ and R′″are independently selected from hydrogen, (C1-C8)alkyl and heteroalkyl,unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C1-C4)alkyl,and (unsubstituted aryl)oxy-(C1-C4)alkyl.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CH2)q-U-, wherein T and U are independently —NH—, —O—, —CH2- ora single bond, and q is an integer of from 0 to 2. Alternatively, two ofthe substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula -A-(CH2)r-B—,wherein A and B are independently —CH2-, —O—, —NH—, —S—, —S(O)—,—S(O)2-, —S(O)2NR′— or a single bond, and r is an integer of from 1 to3. One of the single bonds of the new ring so formed may optionally bereplaced with a double bond. Alternatively, two of the substituents onadjacent atoms of the aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula —(CH2)s-X—(CH2)t-, where s and t areindependently integers of from 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—,—S(O)2-, or —S(O)2NR′—. The substituent R′ in —NR′— and —S(O)2NR′— isselected from hydrogen or unsubstituted (C1-C6)alkyl.

As used herein, the term “heteroatom” is meant to include oxygen (O),nitrogen (N), sulfur (S) and silicon (Si).

The term “pharmaceutically acceptable salts” or “pharmaceuticallyacceptable carrier” is meant to include salts of the active compoundswhich are prepared with relatively nontoxic acids or bases, depending onthe particular substituents found on the compounds described herein.When compounds of the present invention contain relatively acidicfunctionalities, base addition salts can be obtained by contacting theneutral form of such compounds with a sufficient amount of the desiredbase, either neat or in a suitable inert solvent. Examples ofpharmaceutically acceptable base addition salts include sodium,potassium, calcium, ammonium, organic amino, or magnesium salt, or asimilar salt. When compounds of the present invention contain relativelybasic functionalities, acid addition salts can be obtained by contactingthe neutral form of such compounds with a sufficient amount of thedesired acid, either neat or in a suitable inert solvent. Examples ofpharmaceutically acceptable acid addition salts include those derivedfrom inorganic acids like hydrochloric, hydrobromic, nitric, carbonic,monohydrogencarbonic, phosphoric, monohydrogenphosphoric,dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, orphosphorous acids and the like, as well as the salts derived fromrelatively nontoxic organic acids like acetic, propionic, isobutyric,maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic,phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric,methanesulfonic, and the like. Also included are salts of amino acidssuch as arginate and the like, and salts of organic acids likeglucuronic or galactunoric acids and the like (see, e.g., Berge et al.,Journal of Pharmaceutical Science 66:1-19 (1977)). Certain specificcompounds of the present invention contain both basic and acidicfunctionalities that allow the compounds to be converted into eitherbase or acid addition salts. Other pharmaceutically acceptable carriersknown to those of skill in the art are suitable for the presentinvention.

The neutral forms of the compounds may be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents, but otherwise the salts are equivalent to the parentform of the compound for the purposes of the present invention.

In addition to salt forms, the present invention provides compoundswhich are in a prodrug form. Prodrugs of the compounds described hereinare those compounds that readily undergo chemical changes underphysiological conditions to provide the compounds of the presentinvention. Additionally, prodrugs can be converted to the compounds ofthe present invention by chemical or biochemical methods in an ex vivoenvironment. For example, prodrugs can be slowly converted to thecompounds of the present invention when placed in a transdermal patchreservoir with a suitable enzyme or chemical reagent.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are intended to beencompassed within the scope of the present invention. Certain compoundsof the present invention may exist in multiple crystalline or amorphousforms. In general, all physical forms are equivalent for the usescontemplated by the present invention and are intended to be within thescope of the present invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, diastereomers,geometric isomers and individual isomers are all intended to beencompassed within the scope of the present invention.

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present invention, whether radioactive or not, areintended to be encompassed within the scope of the present invention.

Assays for Thio Semicarbazone and Semicarbazone Inhibitors of CysteineProteases

The compounds of the invention are screened for effectiveness againstcathepsin-L like cysteine proteases in vitro and for effectiveness indisrupting the infectious life cycle of a parasite or malignancypotential of a cancer cell in cell culture and in vivo disease modelsystems.

For in vitro cysteine protease inhibition determinations, a compound'seffectiveness can be given by an IC50 value. In these assays, the enzymeto be inhibited (e.g., a cruzain or cruzipain, a rhodesain, a brucipain,a congopain, a falcipain, CPB2.8 Delta CTE, a cathepsin-L, cathepsin-B,a cathepsin-H, a cathepsin-K, a cathepsin-S) is first incubated withvarying concentrations (about 20-50,000 nM) of a test compound. To thisis added a short peptide substrate of the enzyme of 1 to 10 amino acids,usually a di- or tri-peptide substrate, which is labeled with either afluorogenic or chromogenic moiety. An exemplary chromogenic moiety isp-nitro-anilide (pNA). Fluorogenic labels are generally comprised of afluorescent donor, such as ortho-aminobenzoic acid (Abz) orbenzyloxycarbonyl (Z), and a fluorescent quencher, such as7-(4-methyl)-coumarylamide (AMC), methyl-7-aminocoumarin amide (MCA),7-amino-4-trifluoromethylcoumarin (AFC) orN-(ethylenediamine)-2,4-dinitrophenyl amide (EDDnp), where the donor andquencher are on either terminus of the peptide substrate. Exemplarypeptide substrates include Phe-Arg, Arg-Arg, Phe-Arg-X (X=Ala, Arg), andPhe-X-Ser-Arg-Gln (X=Arg, 4-aminomethyl-phenylalanine (Amf),4-aminomethyl-N-isopropyl-phenylalanine (Iaf), 4-piperidinyl-alanine(Ppa) or 4-aminocyclohexyl-alanine (Aca)). Cleavage of the labeledsubstrate induces a chromogenic or fluorescent signal that is measuredusing spectrophotometer or a spectrofluorometer, respectively. Signalsinduced in the presence of varying concentrations of test compound aremeasured in comparison to a positive control of enzyme and substrate anda negative control of enzyme in diluent (e.g., DMSO). Spontaneouscleavage of substrate is measured in controls with substrate alone. IC50values are determined graphically using compound inhibitorconcentrations in the linear portion of a plot of inhibition versus log[I]. Inhibition of a target protease is achieved when the IC50 value isless than about 1000 nM, preferably less than about 500, 300 or 100 nM,more preferably less than about 90, 80, 70, 60, 50, 40, 30, 20 or 10 nM.

Anti-parasitic capacities of the compounds can be measured using cellculture assays. Cultured mammalian cells that are susceptible toinfection by a target protozoan, such as for example, macrophages,erythrocytes, lymphocytes, fibroblasts or other cutaneous cells,hepatocytes, cardiocytes or myocytes are infected with infectiousparasitic bodies, such as trypomastigotes to introduce trypanosomeinfection, merozoites to introduce plasmodium infection, orpromastigotes to introduce leishmania infection. The culture medium isreplaced to remove superfluous infectious parasitic bodies and to addtest protease inhibitor compounds. Positive or treated control culturesare given a known parasitic inhibitor. For example, N-methylpiperazine-Phe-homoPhe-vinyl sulfone phenyl (N-Pip-F-hF-VSPh) is knownto inhibit trypanosomes. Negative or untreated control cultures aregiven only diluent (e.g., DMSO) in medium. Cultures are maintained for atime period that encompasses several intracellular cycles of the targetparasite in untreated controls, usually about 30 days, but as long as35, 40, 45 or 50 days or longer, as necessary. Cells are monitored,usually daily but this can be more or less often, for the presence orabsence of parasitic infection, usually by contrast phase microscopy.The comparative effectiveness of each test protease inhibitor compoundis determined from plots of the duration of the intracellular cycle ofthe target parasite in treated versus untreated control cultures(generally measured in days).

The ability of compounds of the present invention to inhibit cellinvasiveness and migration can also be tested using cellular motilityand cellular invasion assays. These assays are particularly applicableto measuring the inhibition of migration of cancer and inflammatorycells whose migration requires, at least in part, the catalytic activityof at least one cysteine protease such as a cathepsin-L, a cathepsin-B,a cathepsin-H, a cathepsin-K or a cathepsin-S. In vitro cellularmotility assays are generally carried out using transwell chambers(available from Corning-Costar), with upper and lower culturecompartments separated by filters, for example, polycarbonate filterswith 8 μm pore size. In vitro cellular invasion assays are conductedusing matrigel precoated filters (for example, 100 μg/cm² matrigel on afilter with 8 μm pore size; available from Becton Dickinson). Prior toinvasion assays, the matrigel matrix is reconstituted with serum-freecell culture medium. Excess media is removed from the filters and achemoattractant is placed in the lower compartment of a transwellchamber, for example 5 μg/ml collagen I can be used for a tumor cell. Aspecified number of cells radiolabeled with ³H-thymidine are seeded ontothe filter in motility assays or onto the reconstituted matrigelbasement membrane for invasion assays. Cells passing through the filtersand attaching to the lower sides of uncoated or matrigel-coated areharvested using trypsin/EDTA, and cell-bound radioactivity is measuredin a liquid scintillation counter. The number of migrating cells iscalculated by measuring the radioactivity of cells on the underside of afilter in comparison to the radioactivity of a parallel culturecontaining an identical number of cells to what was originally seeded onthe top of the filter or matrigel coating.

The ability of the protease inhibitor compounds to prevent or treatparasitic infections or cancer cell or inflammatory cell invasion ormigration in a host subject also can be tested using in vivo diseasemodels. Experimental animal disease models for trypanosomiasis,leishmania, and malaria are known in the art. For example, murine modelsfor trypanosomiasis are disclosed in Duthie and Kahn, J Immunol (2002)168:5778, Mucci, et al, Proc Natl Acad Sci (2002) 99:3896, Zuniga, etal, J Immunol (2002) 168:3965 and in Guarner, et al (2001) Am J Trop MedHyg 65:152. Murine models for leishmania are described in Rhee, et al, JExp Med (2002) 195:1565, and in Hommel, et al, Ann Trop Med Parasitol(1995) 89 Supp 1:55. Murine models of malaria are published in Sanni, etal, Methods Mol Med (2002) 72:57, Renia, et al, Methods Mol Med (2002)72:41, and Li, et al, Med Microbiol Immunol (2001) 189:115. In mouseparasitic disease models, for example, infected mice are administered atest compound of the present invention, and then monitered foramelioration or abatement of infection in comparison to infected, butuntreated control mice. Alternatively, uninfected mice are treated witha test compound and then inoculated with a infectious parasitic body todetermine the capacity of the compound to prevent parasitic infection.Disease models for cancer and inflammation are also well documented inthe published literature. Murine disease models for human cancersrequire immunodeficient mice (reviewed in Bankert, et al, Front Biosci(2002) 7:c44 and in Hann and Balmain, Curr Opin Cell Biol (2001)13:778). Additional animal cancer models are discussed in Bast, et al,Cancer Medicine, 5^(th) Ed., B. C. Decker, Hamilton, Ontario, Canada).

Preparation of Thio Semicarbazone and Semicarbazone Inhibitors ofCysteine Proteases and Cyclized Pyrazoline Analogues

Synthesis of Thio Semicarbazones and Semicarbazones. Thio Semicarbazonesand Semicarbazones can be synthesized according to Scheme 1. Refluxingaldehyde or ketone with a thio semicarbazide generates thiosemicarbazones. For aldehydes, the reaction is usually complete in lessthan 3 h and no acetic acid is required. For ketones, the reaction isusually run overnight with 1% acetic acid. Yields are generally greaterthan 90% except with a few specific ketones such as the 2-substitutedaryl ketones. Synthesis of semicarbazones is done at room temperature. Asodium acetate solution of semicarbazide hydrochloride salt is added tothe ethanol solution of aldehyde or ketone (Pandeya, S. N. et al.,Pharmacol Res., 37:17-22 (1998)). The product usually precipitates outwith good yield.

Synthesis of the Cyclized Pyrazoline Analogues. Cyclized Pyrazolineanalogues can be synthesized according to Scheme 2. The Mannich reactionof various ketones with formaldehyde and dimethylamine hydrochloridegenerates the Mannich base precursor (Wellinga, K. et al., J. Agric.Food. Chem., 25:987-992 (1977)). The reaction is sensitive to both theamount of hydrochloric acid and the amount of solvent present. Thereaction works best when a minimum amount of ethanol and 2 μL ofacid/mmol ketone is applied. The methyl aryl ketones gave high yieldsabove 80%, while the yields for other alkyl aryl ketones in the Mannichreaction were lower in the range of 30-60%. However, the product mixturecontained only unreacted starting material and the Mannich product. Theunreacted starting material was recovered for repetitive use.Condensation and cyclization of the precursor with the thiosemicarbazide generates the cyclized pyrazoline analogues with yieldsbetween 20% and 50% without optimization. The cyclization also works inthe substituted thio semicarbazide but with a lower yield.

Synthesis of the Various C5-Substituted Thio Semicarbazones andPyrazolines. The C5-side chain is introduced through a Grignard reactionof the suitable aldehyde with the Grignard reagent containing theC5-side chain group in 60-97% yield. The resulting alcohol is oxidizedwith PCC in higher than 80% yield. Then thio semicarbazones andpyrazolines can be made through the previous routes. See synthesisschemes in FIG. 13.

Administration and Pharmaceutical Compositions

Pharmaceutically and physiologically acceptable carriers are determinedin part by the particular composition being administered (e.g., nucleicacid, protein, modulatory compounds or transduced cell), as well as bythe particular method used to administer the composition. Accordingly,there are a wide variety of suitable formulations of pharmaceuticalcompositions of the present invention (see, e.g., Remington'sPharmaceutical Sciences, 17th ed., 1989). Suitable methods ofadministration include oral, nasal, rectal, and parenteraladministration. Other delivery methods known to those of skill in theart can be used, e.g., liposomes, microspheres, and the like. Thecompounds of the invention can also be forumulated as prodrugs for easeof delivery.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the packaged nucleic acidsuspended in diluents, such as water, saline or PEG 400; (b) capsules,sachets or tablets, each containing a predetermined amount of the activeingredient, as liquids, solids, granules or gelatin; (c) suspensions inan appropriate liquid; and (d) suitable emulsions. Tablet forms caninclude one or more of lactose, sucrose, mannitol, sorbitol, calciumphosphates, corn starch, potato starch, microcrystalline cellulose,gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearicacid, and other excipients, colorants, fillers, binders, diluents,buffering agents, moistening agents, preservatives, flavoring agents,dyes, disintegrating agents, and pharmaceutically compatible carriers.Lozenge forms can comprise the active ingredient in a flavor, e.g.,sucrose, as well as pastilles comprising the active ingredient in aninert base, such as gelatin and glycerin or sucrose and acaciaemulsions, gels, and the like containing, in addition to the activeingredient, carriers known in the art.

The compound of choice, alone or in combination with other suitablecomponents, can be made into aerosol formulations (i.e., they can be“nebulized”) to be administered via inhalation. Aerosol formulations canbe placed into pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like.

Suitable formulations for rectal administration include, for example,suppositories, which consist of the packaged nucleic acid with asuppository base. Suitable suppository bases include natural orsynthetic triglycerides or paraffin hydrocarbons. In addition, it isalso possible to use gelatin rectal capsules which consist of acombination of the compound of choice with a base, including, forexample, liquid triglycerides, polyethylene glycols, and paraffinhydrocarbons.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.In the practice of this invention, compositions can be administered, forexample, by intravenous infusion, orally, topically, intraperitoneally,intravesically or intrathecally. Parenteral administration, oraladministration, and intravenous administration are the preferred methodsof administration. The formulations of compounds can be presented inunit-dose or multi-dose sealed containers, such as ampules and vials.

Injection solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described. Cellstransduced by nucleic acids for ex vivo therapy can also be administeredintravenously or parenterally as described above.

The pharmaceutical preparation is preferably in unit dosage form. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form. The composition can, if desired, also contain othercompatible therapeutic agents.

In therapeutic use for the treatment of pain, the compounds utilized inthe pharmaceutical method of the invention are administered at atherapeutically or prophylacticlaly effective dose, e.g., the initialdosage of about 0.001 mg/kg to about 1000 mg/kg daily. A daily doserange of about 0.01 mg/kg to about 500 mg/kg, or about 0.1 mg/kg toabout 200 mg/kg, or about 1 mg/kg to about 100 mg/kg, or about 10 mg/kgto about 50 mg/kg, can be used. The dosages, however, may be varieddepending upon the requirements of the patient, the severity of thecondition being treated, and the compound being employed. The doseadministered to a patient, in the context of the present inventionshould be sufficient to effect a beneficial therapeutic response in thepatient over time. The size of the dose also will be determined by theexistence, nature, and extent of any adverse side-effects that accompanythe administration of a particular vector, or transduced cell type in aparticular patient. Determination of the proper dosage for a particularsituation is within the skill of the practitioner. Generally, treatmentis initiated with smaller dosages which are less than the optimum doseof the compound. Thereafter, the dosage is increased by small incrementsuntil the optimum effect under circumstances is reached. Forconvenience, the total daily dosage may be divided and administered inportions during the day, if desired.

The compounds of the invention can be administered in combination withother therapeutic compounds, either in the same pharmaceuticalpreparation, or in separate pharmaceutical preparations. The additionaltherapeutic or prophylactic compounds may be used to treat the samedisease as the compound of the invention, e.g., a parasitic disease, aprotozoan disease, or a cancer, or can be used to treat a second diseaseother than the disease treated by the compound of the invention. One ormore compounds of the invention can be administered in the samepharmaceutical composition.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of noncritical parameters that could be changed or modified toyield essentially similar results.

Example 1 Determination of the Characteristics of the Compounds

Melting points were determined in open capillaries with a Büchi meltingpoint apparatus, B-540 (Switzerland). Infrared spectra were recorded inan Impact 400 spectrophotometer for a representative set of compounds.¹H NMR spectra were obtained with a Varian Inova-400 NMR spectrometer.Unless otherwise noted, all spectra were recorded with DMSO as solvent.Splitting patterns are designated as follows: s, singlet; br s, broadsinglet; d, doublet; t, triplet; q, quartet; m, multiplet. Couplingconstants (J) are given in hertz. Mass spectral analyses are performedat the Mass Spectrometry facility, University of California, SanFrancisco. Elemental analyses are carried out by Atlantic Microlab,Inc., Norcross, Ga. Unless otherwise stated, yields for the reactionsare higher than 90%.

Example 2 General Procedure for the Preparation of Thio Semicarbazones

Aldehyde or ketone (0.5 mmol) and thio semi-carbazide (0.5 mmol) wasadded to a dry flask. The mixture was dissolved in 10 mL of anhydrousMeOH. For ketone, 1% acetic acid (0.1 mL) was also added to thereaction. The reaction was heated to reflux under nitrogen. Thin LayerChromatography (TLC) was used to monitor whether the reaction wascomplete. The reaction time ranged from 3 h for aldehydes to overnightfor ketones. The solvent was removed in vacuo, and the resulting solidwas rinsed or recrystallized.

Example 3 Characteristics of the Thio Semicarbazone Compounds

Data for 4-(Phenylethynyl)thiophene-2-carboxyaldehyde Thio Semicarbazone(1a): mp 197.6-199.6° C.; ¹H NMR δ 7.42 (m, 3H), 7.52 (m, 2H), 7.60 (s,1H), 7.66 (br s, 1H), 7.95 (s, 1H), 8.19 (br s, 1H), 8.25 (s, 1H), 11.52(s, 1H); HRMS (EI) m/z (M+) calcd for C₁₄H₁₁N₃S₂ 285.0394, found285.0403.

Data for 5-(4-Chlorophenyl)-2-furancarboxaldehyde Thio Semicarbazone(1b): mp 199.2-200.8° C.; ¹H NMR d 7.06 (d, 1H, J=3.2), 7.16 (d, 1H,J=3.6), 7.49 (d, 2H, J=8.4), 7.77 (br s, 1H), 7.85 (d, 2H, J=8.0), 7.96(s, 1H), 8.28 (br s, 1H), 11.50 (s, 1H); HRMS (EI) m/z (M⁺) calcd forC₁₂H₁₀CIN₃OS 279.0233, found 279.0237.

Data for 5-(2-Methoxyphenyl)-2-furancarboxaldehyde Thio Semicarbazone(1c): mp 209.5-211.1° C.; IR (neat, cm⁻¹) 3389, 3234, 3138, 3051, 2983,2930, 2839, 1603, 1545, 1487, 1343, 1299, 1241, 1101, 1019, 923, 845,797, 759; ¹H NMR δ 3.92 (s, 3H), 7.04 (m, 3H), 7.13(d, 1H, J=8.8), 7.33(t, 1H, J=7.8), 7.76 (br s, 1H), 7.94 (d, 1H, J=7.6), 7.97 (s, 1H), 8.24(br s, 1H), 11.46 (s, 1H); HRMS (EI) m/z (M⁺) calcd for C₁₃H₁₃N₃O₂S275.0728, found 275.0720.

Data for2-(1-Methyl-3-trifluoromethyl)pyrazol-5-yl)-thiophene-5-carboxaldehydeThio Semicarbazone (1d): mp 222.4° C. dec; ¹H NMR δ 4.05 (s, 3H), 7.06(s, 1H), 7.52 (m, 2H), 7.62 (br s, 1H), 8.23 (s. 1H), 8.28 (br s, 1H),11.57 (s, 1H); HRMS (EI) m/z (M⁺) calcd for C₁₁H₁₀F₃N₅S₂ 333.0330, found333.0337.

Data for 5-(3-Chlorophenyl)-2-furancarboxaldehyde Thio Semicarbazone(1e): mp 198.8-199.8° C.; ¹H NMR δ 7.07 (d, 1H, J=3.6), 7.24 (d, 1H,J=3.6), 7.38 (d, 1H, J=8.0), 7.46 (m, 1H), 7.79 (d, 1H, J=8.0), 7.83 (brs, 1H), 7.89 (d, 1H, J=1.6), 7.96 (s, 1H), 8.28 (br s, 1H), 11.51 (s,1H); HRMS (EI) m/z (M⁺) calcd for C₁₂H₁₀CIN₃OS 279.0233, found 279.0231.

Data for 5-(4-Bromophenyl)-2-furancarboxaldehyde Thio Semicarbazone(1f): ¹H NMR (isomer ratio 3:1) (major isomer) δ 7.14 (dd, 1H, J=1,2,3,6), 7.29 (d, 1H, J=1.2), 7.30 (s, 1H), 7.72 (m, 2H), 7.77 (m, 2H),8.14 (br s, 1H), 8.61 (br s, 1H), 10.53 (s, 1H); (minor isomer) a 7.06(d, 1H, J=2.4), 7.17 (m, 1H), 7.62 (d, 2H, J=7.6), 7.77 (m, 3H), 7.96(s, 1H), 8.28 (br s, 1H), 11.50 (s, 1H); HRMS (EI) m/z (M⁺) calcd forC₁₂H₁₀BrN₃OS 322.9728, found 322.9723.

Data for 1-(Phenylsulfonyl)-2-pyrrolecarboxaldehyde Thio Semicarbazone(1 g): mp dec upon heating; ¹H NMR δ 6.44 (t, 1H, J=3,4), 7.10 (d, 1H,J=2.0), 7.55 (s, 1H), 7.68 (t, 2H, J=7.8), 7.77 (t, 1H, J=7.4), 7.81 (brs, 1H), 7.96 (d, 2H, J=8.0), 8.17 (br s, 1H), 8.49 (s, 1H), 11.49 (s,1H); HRMS (EI) m/z (M+) calcd for C₁₂H₁₂N₄O₂S₂ 308.0402, found 308.0407.

Data for 4′-Morpholinoacetophenone Thio Semicarbazone (1 h): mp231.2-232.8° C.; ¹H NMR δ 2.23 (s, 3H), 3.16 (t, 4H, J=4.8), 3.73 (t,4H, J=4.8), 6.90 (d, 2H, J=8.8), 7.79 (d, 2H, J=8.8), 7.80 (s, 1H), 8.14(br s, 1H), 10.04 (s, 1H); HRMS (EI) m/z (M⁺) calcd for C₁₃H₁₈N₄OS278.1201, found 278.1210.

Data for 3′-Bromopropiophenone Thio Semicarbazone (1i): mp 144.3-144.1°C.; IR (neat, cm⁻¹) 3413, 3238, 3144, 3059, 2974, 2920, 1584, 1509,1470, 1420, 1280, 1115, 1051, 857, 787; ¹H NMR δ 0.99 (t, 3H, J=7.4),2.85 (q, 2H, J=7.6), 7.33 (t, 1H, J=8.0), 7.55 (d, 1H, J=8.0), 7.85 (d,1H, J=7.6), 8.04 (br s, 1H), 8.13 (s, 1H), 8.26 (br s, 1H), 10.30 (s,1H); HRMS (EI) m/z (M⁺) calcd for C₁₀H₁₂BrN₃S 284.9935, found 284.9936.Anal. (C₁₀H₁₂BrN₃S)C, H, Br, N, S.

Data for 3-Hydroxy-4-methoxybenzaldehyde Thio Semicarbazone (1j): mp175.3-177.5° C.; ¹H NMR δ 3.79 (s, 3H), 6.92 (d, 1H, J=8.4), 7.10 (dd,1H, J=1.6, 8.4), 7.24 (d, 1H, J=2.0), 7.78 (br s, 1H), 7.90 (s, 1H),8.06 (br s, 1H), 9.03 (s, 1H), 11.24 (s, 1H); HRMS (EI) m/z (M⁺) calcdfor C₉H₁₁N₃O₂S 225.0572, found 225.0566.

Data for 6′-Methoxy-2′-propiononaphthone Thio Semicarbazone (1k): mp170.5-171.2° C.; ¹H NMR δ 1.07 (t, 3H, J=7.4), 2.96 (q, 2H, J=7.4), 3,87(s, 3H), 7.17 (dd, 1H, J=2.4, 8.8), 7.33 (d, 1H, J=2.4), 7.77 (d, 1H,J=8.8), 7.89 (d, 1H, J=8.8), 7.98 (br s, 1H), 8.19 (d, 1H, J=8.8), 8.26(s, 1H), 8.30 (br s, 1H), 10.32 (s, 1H); HRMS (EI) m/z (M⁺) calcd forC₁₅H₁₇N₉OS 287.1092, found 287.1090.

Data for 4-Dimethylamino-1-naphthaldehyde Thio Semicarbazone (1l): mp170.5-171.2° C.; ¹H NMR δ 2.87 (s, 6H), 7.10 (d, 1H, J=8.0), 7.55 (m,1H), 7.61 (m, 1H), 7.83 (br s, 1H), 8.03 (d, 1H, J=8.0), 8.18 (m, 2H),8.42 (d, 1H, J=8.4), 8.78 (s, 1H), 11.32 (s, 1H); HRMS (EI) m/z (M⁺)calcd for C₁₄H₁₆N₄S 272.1096, found 272.1094.

Data for 1-Methylindole-3-carboxaldehyde Thio Semicarbazone (1m): mp200.3-201.2° C.; ¹H NMR δ 3.80 (s, 3H), 7.15 (t, 1H, J=7.4), 7.25 (t,1H, J=7.6), 7.40 (br s, 1H), 7.47 (d, 1H, J=8.0), 7.79 (s, 1H), 8.00 (brs, 1H), 8.22 (d, 1H, J=7.6), 8.26 (s, 1H), 11.13 (s, 1H); HRMS (EI) m/z(M+) calcd for C₁₁H₁₂N₄S 232.0783, found 232.0786.

Data for 2-Methoxy-1-naphthaldehyde Thio Semicarbazone (1n): mp156.2-157.6° C.; ¹H NMR δ 3.96 (s, 3H), 7.40 (t, 1H, J=7.4), 7.48 (d,1H, J=9.2), 7.50 (br s, 1H), 7.56 (t, 1H, J=7.8), 7.88 (d, 1H, J=8.0),8.02 (d, 1H, J=9.2), 8.20 (br s, 1H), 8.80 (s, 1H), 8.92 (d, 1H, J=8.8),11.48 (s, 1H); HRMS (EI) m/z (M⁺) calcd for C₁₃H₁₃N₃OS 259.0779, found259.0772.

Data for 3′-Chloropropiophenone Thio Semicarbazone (2a): mp 136.6-138.3°C.; IR (neat, cm⁻¹)3408, 3203, 3155, 2970, 2941, 1598, 1511, 1472, 1418,1301, 1233, 1112, 1053, 1004, 859, 791; ¹H NMR δ 0.98 (t, 3H, J=7.4),2.86 (q, 2H, J=7.6), 7.41 (m, 2H), 7.82 (m, 1H), 8.03 (s, 1H), 8.08 (brs, 1H), 8.31 (brs, 1H), 10.36 (s, 1H); HRMS (EI) m/z (M⁺) calcd forC₁₀H₁₂CIN₃S 241.0440, found 241.0449.

Data for 3′-Trifluoromethylpropiophenone Thio Semicarbazone (2b): mp156.0-157.9° C.; IR (neat, cm⁻¹) 3427, 3272, 3155, 3053, 2975, 2941,1618, 1525, 1481, 1345, 1306, 1189, 1146, 1078, 864, 810, 703; ¹H NMR δ1.00 (t, 3H, J=7.4), 2.92 (q, 2H, J=7.4), 7.61 (t, 1H, J=7.8), 7.72 (d,1H, J=7.6), 8.11 (br s, 1H), 8.20 (s, 2H), 8.35 (br s, 1H), 10.42 (s,1H); HRMS (EI) m/z (M⁺) calcd for C₁₁H₁₂F₃N₃S 275.0704, found 275.0709.Anal. (C₁₁H₁₂F₃N₃S)C, H, F, N, S.

Data for 3′-Fluoropropiophenone Thio Semicarbazone (2c): mp 138.8-139.5°C.; ¹H NMR δ 0.99 (t, 3H, J=7.6), 2.86 (q, 2H, J=7.6), 7.20 (dt, 1H,J=2.4, 8.6), 7.43 (m, 1H), 7.69 (d, 1H, J=8.0), 7.87 (d, 1H, J=11.2),8.07 (br s, 1H), 8.31 (br s, 1H), 10.36 (s, 1H); HRMS (EI) m/z (M⁺)calcd for C₁₀H₁₂FN₃S 225.0736, found 225.0738.

Data for Propiophenone Thio Semicarbazone (2d): mp 116.8-117.9; ¹H NMR δ1.01 (t, 3H,J=7.6), 2.86 (q, 2H,J=7.6), 7.38 (m, 3H), 7.89 (m, 3H), 8.22(brs, 1H), 10.27 (s, 1H); HRMS (EI) m/z (M⁺) calcd for C₁₀H₁₃N₃S207.0830, found 207.0826.

Data for 4′-Bromopropiophenone Thio Semicarbazone (2e): mp 171.3-173.0°C.; ¹H NMR δ 0.98 (t, 3H, J=7.4), 2.84 (q, 2H, J=7.4), 7.55 (d, 2H,J=8.4), 7.88 (d, 2H, 8.4), 7.97 (br s, 1H), 8.31 (br s, 1H), 10.38 (s,1H); HRMS (EI) m/z (M⁺) calcd for C₁₀H₁₂BrN₃S 284.9935, found 284.9938.

Data for 4′-Chloropropiophenone Thio Semicarbazone (2f): yield 60%; mp175.1-176.8° C.; ¹H NMR δ 0.99 (t, 3H, J=7.6), 2.84 (q, 2H, J=7.6), 7.41(d, 2H, J=8.8), 7.95 (d, 2H, J=8.4), 7.96 (br s, 1H), 8.30 (br s, 1H),10.37 (s, 1H); HRMS (EI) m/z (M⁺) calcd for C₁₀H₁₂CIN₃S 241.0440, found241.0447.

Data for 4′-Methoxypropiophenone Thio Semicarbazone (2g): mp117.8-119.8° C.; ¹H NMR δ 0.99 (t, 3H, J=7.6), 2.83 (q, 2H, J=7.6), 3.78(s, 3H), 6.92 (d, 2H, J=9.2), 7.85 (br s, 1H), 7.86 (d, 2H, J=8.8), 8.19(br s, 1H), 10.21 (s, 1H); HRMS (EI) m/z (M⁺) calcd for C₁₀H₁₅N₃OS237.0936, found 237.0933.

Data for 3′,5′-bis(trifluoromethyl)propiophenone Thio Semicarbazone(2h): mp 188.0-190.2° C.; IR (neat, cm⁻¹) 3245, 3153, 2982, 2936, 1596,1508, 1466, 1392, 1281, 1171, 1126, 893, 866; ¹H NMR δ 0.99 (t, 3H,J=7.6), 2.98 (q, 2H, t, 3H, J=7.6), 8.07 (s, 1H), 8.33 (br s, 1H), 8.43(br s, 1H), 8.49 (s, 2H), 10.51 (s, 1H); HRMS (EI) m/z (M+H⁺) calcd forC₁₂H₁₂F₆N₃S 344.0656, found 344.0671.

Data for 3′,4′-Dichloropropiophenone Thio Semicarbazone (2i): mp185.6-186.4° C.; IR (neat, cm⁻¹) 3420, 3249, 3150, 3041, 2980, 2942,1603, 1513, 1475, 1390, 1300, 1101, 1063, 888, 860, 793; ¹H NMR δ 0.98(t, 3H, J=7.6), 2.86 (q, 2H. J=7.6), 7.61 (d, 1H, J=8.8), 7.85 (dd, 1H,J=2.0, 8.6), 8.12 (br s, 1H), 8.22 (d, 1H, J=2.0) 8.30 (br s, 1H), 10.35(s, 1H); FIRMS (EI) m/z (M⁺) calcd for C₁₀H₁₁C₁₂N₃S 275.0051, found275.0053.

Data for 2′,4′-Dichloropropiophenone Thio Semicarbazone (2j): mp171.8-173.0° C.; ¹H NMR δ 1.04 (t, 3H, J=7.6), 2.49 (q, 2H, J=7.6), 7.33(d, 1H, J=8.4), 7.53 (dd, 1H, J=2.0, 8.4), 7.75 (d, 1H, J=2.0), 7.77 (brs, 1H), 8.26 (br s, 1H), 9.42 (s, 1H); HRMS (EI) m/z (M⁺) calcd forC₁₀H₁₁C₁₂N₃S 275.0051, found 275.0049.

Data for 3′-Methoxyacetophenone Thio Semicarbazone (2k): mp 195.1-196.7°C.; ¹H NMR δ 2.28 (d, 3H, J=3.2), 3.79 (d, 3H, J=2.8), 6.95 (d, 1H,J=8.0), 7.29 (dt, 1H, J=2.8, 8.0), 7.44 (m, 2H), 7.88 (br s, 1H), 8.21(br s, 1H), 10.12 (s, 1H); HRMS (EI) m/z (M⁺) calcd for C₁₀H₁₃N₃OS223.0779, found 223.0777.

Data for 2′-Bromoacetophenone Thio Semicarbazone (2l): yield 40%; mp144.5-146.2° C.; ¹H NMR δ 2.27 (s, 3H), 7.31 (m, 1H), 7.42 (m, 2H), 7.60(br s, 1H), 7.65 (d, 1H, J=8.0), 8.24 (br s, 1H), 10.34 (s, 1H); HRMS(EI) m/z (M⁺) calcd for C₉H₁₀BrN₃S 270.9779, found 270.9775.

Data for 2′-Chloroacetophenone Thio Semicarbazone (2m): yield 45%; mp155.8-156.7° C.; ¹H NMR δ 2.28 (s, 3H), 7.38 (m, 2H), 7.49 (m, 2H), 7.62(br s, 1H), 8.24 (br s, 1H), 10.34 (s, 1H); HRMS (EI) m/z (M⁺) calcd forC₉H₁₀CIN₃S 227.0284, found 227.0289.

Data for 2,3-Dichlorobenzaldehyde Thio Semicarbazone (2n): mp232.3-233.6° C.; ¹H NMR δ 7.37 (t, 1H, J=8.0), 7.65 (m, 1H), 8.17 (br s,1H), 8.29 (d, 1H, J=8.0), 8.35 (br s, 1H), 8.48 (s, 1H), 11.67 (s, 1H);HRMS (EI) m/z (M⁺) calcd for C₈H₇Cl₂N₃S 246.9738, found 246.9730.

Data for 3,5-Dichlorobenzaldehyde Thio Semicarbazone (2o): mp234.1-235.5° C.; ¹H NMR δ 7.57 (t, 1H, J=1.6), 7.93 (d, 2H, J=1.6), 7.96(s, 1H), 8.31 (br s, 1H), 11.59 (s, 1H); HRMS (EI) m/z (M⁺) calcd forC₈H₇Cl₂N₃S 246.9738, found 246.9737.

Data for 2-Trifluoromethylbenzaldehyde Thio Semicarbazone (2p): mp248.3-249.4° C.; ¹H NMR δ 7.58 (t, 1H, J=7.6), 7.68 (t, 1H, J=7.6), 7.75(d, 1H, J=8.0), 8.15 (br s, 1H), 8.35 (br s, 1H), 8.43 (s, 1H), 8.50 (d,1H, J=8.0), 11.67 (s, 1H); HRMS (EI) m/z (M⁺) calcd for C₉H₈F₃N₃S247.0391, found 247.0391.

Data for 3-Bromobenzaldehyde Thio Semicarbazone (3a): mp 200.6-210.9°C.; IR (neat, cm⁻¹) 3388, 3233, 3149, 3024, 2984, 2805, 1604, 1534,1467, 1355, 1310, 1220, 1101, 941, 897, 832, 792; ¹H NMR δ 7.34 (t, 1H,J=7.6), 7.55 (d, 1H, J=7.2), 7.68 (d, 1H, J=7.6), 7.99 (s, 1H), 8.18 (s,2H), 8.24 (br s, 1H), 11.49 (s, 1H); HRMS (EI) m/z (M⁺) calcd forC₈H₈BrN₃S 256.9622, found 256.9618.

Data for 3′-Bromoacetophenone Thio Semicarbazone (3b): mp 174.2-174.9°C.; IR (neat, cm⁻¹) 3428, 3263, 3144, 3059, 1604, 1524, 1310, 1116, 871,802, 732; ¹H NMR δ 2.27 (s, 3H), 7.33 (t, 1H, J=8.0), 7.55 (dd, 1H,J=1.0, 8.0), 7.88 (d, 1H, J=8.0), 8.10 (br s, 1H), 8.18 (s, 1H), 8.31(br s, 1H), 10.22 (s, 1H); HRMS (EI) m/z (M⁺) calcd for C₉H₁₀BrN₃S270.9779, found 270.9778.

Data for 3-Trifluoromethylbenzaldehyde Thio Semicarbazone (3c): mp220.7-221.7° C.; ¹H NMR δ 7.62 (t, 1H, J=7.6), 7.71 (d, 1H, J=8.0), 8.02(d, 1H, J=7.6), 8.10 (s, 1H), 8.25 (br s, 1H), 8.26 (s, 1H), 8.28 (br s,1H), 11.55 (s, 1H); HRMS (EI) m/z (M⁺) calcd for C₉H₈F₃N₃S 247.0391,found 247.0395.

Data for 3-Trifluoromethylacetophenone Thio Semicarbazone (3d): mp197.9-201.2° C.; ¹H NMR δ 2.33 (s, 3H), 7.60 (t, 1H, J=7.6), 7.72 (d,1H, J=7.6), 8.13 (br s, 1H), 8.22 (m, 2H), 8.34 (br s, 1H), 10.29 (s,1H); HRMS (EI) m/z (M⁺) calcd for C₁₀H₁₀F₃N₃S 261.0548, found 261.0545.

Data for 3,5-Bis(trifluoromethyl)benzaldehyde Thio Semicarbazone (3e):mp 229.8-230.3° C.; ¹H NMR δ 8.04 (s, 1H), 8.16 (s, 1H), 8.38 (br s, 1H8.46 (br s, 1H 8.54 (s, 2H), 11.71 (s, 1H); HRMS (EI) m/z (M⁺) calcd forC₁₀H₇F₆N₃S 315.0265, found 315.0269.

Data for 3′,5′-Bis(trifluoromethyl)acetophenone Thio Semicarbazone (3f):mp 238° C. dec; ¹H NMR δ 2.38 (s, 3H), 8.06 (s, 1H), 8.36 (br s, 1H),8.42 (br s, 1H), 8.53 (s, 2H), 10.37 (s, 1H); HRMS (EI) m/z (M⁺) calcdfor C₁₁H₉F₆N₃S 329.0421, found 321.0429.

Data for 3,4-Dichlorobenzaldehyde Thio Semicarbazone (3g): ¹H NMR δ 7.64(d, 1H, J=8.4), 7.71 (dd, 1H, J=1.8, 8.4), 7.98 (s, 1H), 8.24 (s, 1H),8.27 (br s, 1H), 11.55 (s, 1H); HRMS (EI) m/z (M⁺) calcd for C₈H₇C₁₂N₃S246.9738, found 246.9732.

Data for 3′,4′-Dichloroacetophenone Thio Semicarbazone (3h): mp196.0-197.9° C.; ¹H NMR δ 2.27 (s, 3H), 7.61 (d, 1H, J=8.8), 7.88 (dd,1H, J=0.8, 8.4), 8.17 (br s, 1H), 8.27 (s, 1H), 8.34 (br s, 1H), 10.27(s, 1H); HRMS (EI) m/z (M⁺) calcd for C₉H₉Cl₂N₃S 260.9894, found260.9891. Anal. (C₉H₉—Cl₂N₃S)C, H, Cl, N, S.

Data for 2-Acetyl-5-bromothiophene Thio Semicarbazone (3i): mp205.1-207.3° C.; ¹H NMR δ 2.27 (s, 3H), 7.19 (m, 1H), 7.32 (d, 1H,J=3.6), 7.49 (br s, 1H), 8.31 (br s, 1H), 10.37 (s, 1H); HRMS (EI) m/z(M⁺) calcd for C₇H₈BrN₃S₂ 276.9343, found 276.9349.

Data for 2-Acetyl-5-chlorothiophene Thio Semicarbazone (3j): mp237.1-238.2° C.; ¹H NMR δ 2.27 (s, 3H), 7.09 (d, 1H, J=4.0), 7.36 (d,1H, J=4.0), 7.48 (br s, 1H), 8.31 (br s, 1H), 10.38 (s, 1H); HRMS (EI)m/z (M⁺) calcd for C₇H₈CIN₃S₂ 232.9848, found 232.9845.

Example 4 General Procedure for Synthesizing Cyclized PyrazolineAnalogues of Thio Semicarbazones

Mannich Reaction. A 20 μL portion of concentrated hydrochloric acid wasadded to a mixture of ketone (10 mmol), paraformaldehyde (13 mmol, 390mg), and dimethylamine hydrochloride (13 mmol, 1.059 g) in 5 mL ofethanol. The reaction was refluxed overnight under nitrogen. In somecases, precipitates formed and the product was obtained by filtration inethanol. If no precipitate was formed, the solvent was removed. A fewdrops of HCl is added, and the mixture is worked up with dichloromethaneand water. The dichloromethane layer was discarded. The aqueous layerwas adjusted to basic and extracted with dichloromethane (3×). Thedichloromethane layer was combined and dried. The product was obtainedby removal of dichloromethane.

Cyclization. Thio semicarbazide or substituted thio semicarbazide (0.5mmol) was dissolved in MeOH (5 mL) upon refluxing under nitrogen. Sodiumhydroxide (50%) (0.18 mL) was added to the reaction. A warm methanol (5mL) solution of the previous Mannich reaction product (0.5 mmol) wasthen added dropwise to the reaction mixture. After the reaction wasrefluxed for 1 h, methanol was removed at reduced pressure. The residuewas dissolved in dichloromethane and washed with water. Evaporation ofthe solvent and purification through chromatography gave the cyclizedanalogues.

Example 5 Characteristics of the Cyclized Pyrazoline Analogues

Data for 3-(3-Bromophenyl)-2-pyrazoline-1-thiocarboxamide (4a): Mannichyield 84%, cyclization yield 24%; mp 172.9-174.5° C., ¹H NMR δ 3.28 (t,2H, J=10.0), 4.14 (t, 2H, J=10.0), 7.41 (t, 1H, J=8.0), 7.64 (d, 1H,J=8.4), 7.75 (d, 1H, J=8.0), 7.89 (br s, 1H), 8.00 (br s, 1H), 8.13 (s,1H); HRMS (EI) m/z (M⁺) calcd for C₁₀H₁₀BrN₃S 282.9779, found 282.9780.

Data for 3-(3-Bromophenyl)-4-methyl-2-pyrazoline-1-thiocarboxamide (4b):Mannich yield 81%, cyclization yield 62%; mp 107.4-109.9° C.; IR (neat,cm⁻¹) 3430, 3240, 3139, 3064, 2954, 2924, 2859, 1589, 1497, 1466, 1366,1086, 1006, 901, 790; ¹H NMR δ 1.17 (d, 3H, J=7.2), 3.80 (m, 1H), 3.92(dd, 1H, J=4.2, 11.6), 4.19 (t, 1H, J=11.6), 7.41 (t, 1H, J=8.0), 7.63(m. 1H), 7.78 (d. 1H, 8.0), 7.94 (br s, 1H), 8.07 (br s, 1H), 8.16 (m,1H); HRMS (EI) m/z (M⁺) calcd for C₁₁H₁₂BrN₃S 296.9935, found 296.9926.

Data for 3-(3-Chlorophenyl)-2-pyrazoline-1-thiocarboxamide (4c): Mannichyield 87%, cyclization yield 25%; mp 154.3-157.0° C.; ¹H NMR δ 3.28 (t,2H, J=9.2), 4.14 (t, 2H, J=10.0), 7.49 (m, 2H), 7.72 (d, 1H, J=7.2),7.89 (br s, 1H), 7.99 (s, 2H); HRMS (EI) m/z (M⁺) calcd for C₁₀H₁₀CIN₃S239.0284, found 239.0284.

Data for 3-(3-Chlorophenyl)-4-methyl-2-pyrazoline-1-thiocarboxamide(4d): Mannich yield 44%, cyclization yield 47%; mp 131.3° C. dec; IR(neat, cm⁻¹) 3432, 3267, 3147, 3062, 2967, 2927, 2881, 1584, 1499, 1464,1364, 1118, 1008, 908, 803, 743; ¹H NMR δ 1.17 (d, 3H, J=7.2), 3.80 (m,1H), 3.94 (dd, 1H, J=4.4, 11.6), 4.19 (t, 1H, J=11.2), 7.50 (m, 2H),7.75 (d, 1H, J=6.8), 7.93 (br s, 1H), 8.03 (s, 1H), 8.07 (br s, 1H);HRMS (EI) m/z (M⁺) calcd for C₁₁H₁₂CIN₃S 253.0440, found 253.0447.

Data for 3-(3,4-Dichlorophenyl)-2-pyrazoline-1-thiocarboxamide (4e):Mannich yield 50%, cyclization yield 20%; mp 199.3-201.4° C.; IR (neat,cm⁻¹) 3427, 3233, 3140, 2921, 2843, 1598, 1501, 1467, 1379, 1102, 888,815; ¹H NMR δ 3.28 (t, 3H, J=10.0), 4.15 (t, 2H, J=10.0), 7.74 (m, 2H),7.94 (br s, 1H), 8.04 (br s, 1H), 8.16 (s, 1H); HRMS (EI) m/z (M⁺) calcdfor C₁₀H₉Cl₂N₃S 272.9894, found 272.9896.

Data for 3-(3,4-Dichlorophenyl)-4-methyl-2-pyrazoline-1-thiocarboxamide(4f): Mannich yield 28%, cyclization yield 52%; mp 171.2-172.7° C.; IR(neat, cm⁻¹) 3434, 3264, 3145, 3051, 2961, 2927, 2868, 1581, 1457, 1368,1129, 1027, 907, 809; ¹H NMR δ 1.17 (d, 3H, J=7.2), 3.80 (m, 1H), 3.95(dd, 1H, J=4.0, 11.6), 4.19 (t, 1H, J=11.6), 7.72 (d, 1H, J=8.0), 7.79(d, 1H, J=8.4), 7.99 (br s, 1H), 8.10 (br s, 1H), 8.21 (s, 1H); HRMS(EI) m/z (M⁺) calcd for C₁₁H₁₁C₁₂N₃S 287.0051, found 287.0052.

Data for 3-(3-Trifluoromethylphenyl)-2-pyrazoline-1-thiocarboxamide(4g): Mannich yield 61%, cyclization yield 30%; mp 159.2-161.1° C.; ¹HNMR δ 3.34 (t, 2H, J=10.4), 4.17 (t, 2H, J=10.0), 7.70 (t, 1H, J=7.6),7.81 (d, 1H, J=8.0), 8.03 (m, 3H), 8.26 (s, 1H); HRMS (EI) m/z (M⁺)calcd for C₁₁H₁₀F₃N₃S 273.0548, found 273.0542.

Data for3-(3-Trifluoromethylphenyl)-4-methyl-2-pyrazoline-1-thiocarboxamide(4h): Mannich yield 40%, cyclization yield 35%; mp 120.8-121.9° C.; IR(neat, cm⁻¹) 3443, 3264, 3145, 3068, 2966, 2932, 2872, 1589, 1487, 1351,1129, 907, 805; ¹H NMR δ 1.19 (d, 3H, J=6.8), 3.88 (m, 1H), 3.97 (dd,1H, J=4.4, 11.2), 4.22 (t, 1H, J=11.2), 7.69 (t, 1H, J=7.6), 7.80 (d,1H, J=7.6), 8.02 (br s, 1H), 8.08 (d, 1H, J=8.4), 8.11 (br s, 1H), 8.30(s, 1H); HRMS (EI) m/z (M⁺) calcd for C₁₂H₁₂F₃N₃S 287.0704, found287.0701. Anal. (C₁₂H₁₂F₃N₃S)C, H, F, N, S.

For N1-amino-substituted compounds, only the data of the representativecompounds are given.

Data for 3′-Bromopropiophenone N-Methyl Thio Semicarbazone (5a): yield61%, with no acetic acid added during reaction; mp 156.6-158.0° C.; IR(neat, cm⁻¹) 3367, 3297, 3197, 3057, 2967, 2932, 1544, 1494, 1469, 1234,1118, 1048, 793; ¹H NMR δ 0.98 (t, 3H, J=7.6); 2.87 (q, 2H, J=7.2), 3.03(s, 3H), 7.36 (t, 1H, J=8.0), 7.57 (d, 1H, J=8.0), 7.87 (d, 1H, J=7.6),8.11 (s, 1H), 8.53 (br s, 1H), 10.39 (s, 1H); HRMS (EI) m/z (M⁺) calcdfor C₁₁H₁₄BrN₃S 299.0092, found 299.0091.

Data for 3-(3-Bromophenyl)-2-pyrazoline-1-(N-methyl)thiocarboxamide(5b): cyclization yield 10%; ¹H NMR δ 2.98 (d, 3H, J=4.4), 3.26 (t, 2H,J=9.6), 4.15 (t, 2H, J=10.0), 7.43 (t, 1H, J=8.0), 7.64 (d, 1H, J=8.4),7.74 (d, 1H, J=7.6), 8.13 (s, 1H), 8.48 (d, 1H, J=4.4); HRMS (EI) rdz(M⁺) calcd for C₁₁H₁₂BrN₃S 296.9935, found 296.9939.

Data for 3′-Bromopropiophenone N-(4-Trifluoromethylphenyl) ThioSemicarbazone (5c): mp 153.7-155.7° C.; IR (neat, cm⁻¹) 3302, 3210,2975, 2935, 1592, 1536, 1463, 1325, 1281, 1115, 840; ¹H NMR δ 1.04 (t,3H, J=7.2), 2.95 (q, 2H, J=7.2), 7.38 (t, 1H, J=8.0), 7.61 (d, 1H,J=8.0), 7.72 (d, 2H, J=8.8), 7.86 (m, 2H), 7.94 (d, 1H, J=8.0), 8.17 (s,1H), 10.30 (s, 1H), 10.98 (s, 1H); HRMS (EI) m/z (M−H) calcd forC₁₇H₁₅Br⁸¹F₃N₃S 430.0023, found 430.0009.

Data for3-(3-Bromophenyl)-2-pyrazoline-1-(IL3-chlorophenyl)thiocarboxamide (5d):yield 35%; mp 197.5-198.8° C.; IR (neat, cm⁻¹) 3337, 3081, 2970, 1606,1559, 1491, 1444, 1363, 1133, 878; ¹H NMR δ 3.36 (t, 2H, J=10.0), 4.25(t, 2H, J=10.0), 7.19 (dd, 1H, J=1.2, 8.0), 7.38 (t, 1H, J=8.0), 7.45(t, 1H, J=8.0), 7.58 (d, 1H, 8.0), 7.68 (dd, 1H, J=1.2, 8.0), 7.73 (t,1H, J=1.6), 7.86 (d, 1H, J=8.0), 8.26 (t, 1H, J=1.6), 10.19 (s, 1H);HRMS (EI) m/z (M⁺) calcd for C₁₆H₁₃Br⁸¹CIN₃S 394.9682, found 394.9694.

Data for3-(3-Chlorophenyl)-2-pyrazoline-1-(N-3-tri-fluoromethylphenyl)thiocarboxamide(5e): cyclization yield 6%; mp 151.8-152.9° C.; ¹H NMR δ 3.38 (t, 2H,J=9.6), 4.27 (t, 2H, 9.6), 7.56 (m, 4H), 7.83 (d, 1H, J=7.2), 7.95 (t,1H, J=7.2), 8.00 (d, 1H, J=7.6), 8.14 (s, 1H), 10.31 (s, 1H); HRMS (EI)m/z (M⁺) calcd for C₁₇H₁₃CIF₃N₃S 383.0471, found 383.0474.

Data for 3-(3-Bromophenyl)-2-pyrazoline-1-(N-hexyl)-thiocarboxamide(5f): cyclization yield 18%; mp 117.0-118.2° C.; ¹H NMR δ 0.86 (m, 3H),1.27 (m, 6H), 1.56 (m, 2H), 3.25 (t, 2H, J=10.4), 3.51 (q, 2H, J=6.8),4.15 (t, 2H, J=10.4), 7.42 (t, 1H, J=7.6), 7.64 (d, 1H, J=7.6), 7.75 (d,1H, J=7.6), 8.12 (d, 1H, J=1.2), 8.49 (t, 1H, J=5.6); HRMS (EI) m/z (M⁺)calcd for C₁₆H₂₂BrN₃S 367.0718, found 367.0724.

Example 6 General Procedure for the Preparation of Semicarbazones

Semicarbazide hydrochloride (1 mmol) and sodium acetate (1 mmol) weredissolved in 1 mL of distilled water.

The solution was slowly added to an ethanol (5 mL) solution of aldehydeor ketone (1 mmol) and the mixture stirred for 2 h. The resultingprecipitate was filtered, washed with water and methanol, and dried.Only the data of the representative compounds are given.

Example 7 Characteristics of the Semicarbazones

Data for 3′-Bromopropiophenone Semicarbazone (6a): mp 182.4-184.2° C.;IR (neat, cm⁻¹) 3473, 3302, 3209, 3068, 2974, 1713, 1593, 1491, 1419,1142, 1065, 831, 801; ¹H NMR δ 0.97 (t, 3H, J=7.6), 2.71 (q, 2H, J=7.6),6.54 (br s, 2H), 7.31 (t, 1H, J=8.0), 7.51 (m, 1H), 7.79 (d, 1H, J=8.0),8.03 (s, 1H), 9.53 (s, 1H); HRMS (EI) m/z (M⁺) calcd for C₁₀H₁₂—BrN₃O269.0164, found 269.0158.

Data for 3′-Bromoacetophenone Semicarbazone (6b): mp 229.3-231.3° C.; ¹HNMR δ 2.15 (s, 3H), 6.55 (br s, 2H), 7.31 (t, 1H, J=7.6), 7.51 (d, 1H,J=7.6), 7.80 (d, 1H, J=8.4), 8.04 (s, 1H), 9.37 (s, 1H); HRMS (EI) m/z(M⁺) calcd for C₉H₁₀BrN₃O 255.0007, found 255.0000.

Data for 5-(3-Chlorophenyl)-2-furancarboxaldehyde Semicarbazone (6c): mp192.1-194.8° C.; ¹H NMR δ 6.44 (s, 2H), 6.92 (d, 1H, J=3.6), 7.20 (d,1H, J=3.6), 7.36 (dd, 1H, J=1.2, 8.0), 7.45 (t, 1H, J=8.0), 7.75 (d, 1H,J=6.8), 7.85 (s, 1H), 10.33 (s, 1H); HRMS (EI) m/z (M⁺) calcd forC₁₂H₁₀—CIN₃O₂ 263.0462, found 263.0467.

Example 8 Reduction of Thio Semicarbazone 1i to1-(3′-bromophenyl)propylaminothiourea (Synthesis of 7a according toscheme 3)

Thio semicarbazone (100 mg) was dissolved in anhydrous methanol. Sodiumborohydride (excess) was added in small portions every 30 min. Thereaction was heated to reflux under nitrogen. The reaction was stoppedwhen most of the starting material was reacted according to TLC. Thenthe reaction was worked up with saturated ammonium chloride solution andethyl acetate. After concentration, chromatography gave pure 7a in 70%yield: mp 110.4-112.4° C.; IR (neat, cm⁻¹) 3403, 3224, 3144, 3064, 2965,2930, 2870, 1589, 1470, 1270, 1066, 872, 787; ¹H NMR δ 0.64 (t, 3H,J=7.2), 1.43 (m, 1H), 1.68 (m, 1H), 3.74 (m, 1H), 7.25 (t. 1H, J=7.6),7.34 (d, 1H, J=7.6), 7.38 (br s, 1H), 7.43 (d, 1H, J=8.0), 7.57 (br s,1H), 7.61 (s, 1H), 8.51 (s, 1H); HRMS (EI) m/z (M+H+) calcd forC₁₀H₁₄BrN₃S 288.0170, found 288.0159.

Example 9 Protease Inhibition Assay

Methods

IC₅₀ Determinations. Inhibitors were screened for effectiveness againstthe T. cruzi cathepsin L-like protease (cruzain) using purifiedrecombinant protein. Cruzain (1 nM) was incubated with 20-50000 nMinhibitor in 100 mM sodium acetate-5 mM DTT buffer (pH 5.50, buffer A)for 5 min at room temperature. A 200 μL portion of Z-Phe-Arg-AMC(Bachem, K_(m)=1 μM) was added to the enzyme-inhibitor reaction to givea 20 μM substrate concentration. The increase in fluorescence(excitation at 355 nM and emission at 460 nM) was followed with anautomated microtiter plate spectrofluorimeter (Molecular Devices,spectraMAX Gemini). Inhibitor stock solutions were prepared at 20 mM inDMSO, and serial dilutions were made in DMSO (0.7% DMSO in assay).Controls were performed using enzyme alone and enzyme with DMSO. IC₅₀values were determined graphically using inhibitor concentrations in thelinear portion of a plot of inhibition versus log [I] (sevenconcentrations were tested, and at least two were in the linear range).

The time dependence of inhibition was determined by incubating cruzainwith inhibitors, DMSO, or enzyme alone at room temperature for timepoints between 90 s and 24 h. The enzymatic activity was determined asabove.

Results

Thio Semicarbazones inhibit cruzain cysteine protease activity.

A library of Parke-Davis chemical compounds was screened. Forty-fivecompounds were identified as active against cruzain. We observed asemicarbazone scaffold common to several of the inhibitors (FIG. 1). Tofurther explore the activity of the thio semicarbazone scaffold aninitial group of 14 compounds were synthesized. Studies of noncovalentinhibitors of curzain, including bisaryl acylhydrazides and ureas,demonstrate that the most active compounds have a six-membered phenylring-five-membered heteroaromatic ring combination (6-5) in the arylposition. This feature was incorporated into some of the compounds. Someother alternatives in the aryl position were also tried including fusedaromatic rings or single aromatic rings. Four of the 6-5 compounds (1b,1c, 1e, 1f) have IC50 values below 300 nM. Two of the alternativecompounds; (1i, 1n) were active at 100 and 560 nM against the enzyme,respectively (FIG. 2).

Structure-Activity Relationship (SAR) Studies

Aryl Group Activity (FIG. 3).

A series of 1i analogues with various substituents on the aryl moietywere synthesized. Compound 2d did not have any substitution on the arylring. Its activity dropped at least 2 orders of magnitude compared tothat of 1i (samples with IC50 values greater than 10 μM were not testedfurther). C-2 substitution with bromine, chlorine, or trifluoromethylgroups resulted in poor activity. Though the compounds in FIG. 2 vary atC-5 (ethyl, methyl, or hydrogen), the differences in IC50 resulting fromthe C-5 differences are not significant compared to those resulting fromthe differences in the phenyl ring substitution as indicated by the SARshown in FIG. 11. Among the C-3 variants, the trifluoromethylsubstitution (2b) resulted in a better inhibitor compared to 1i with anIC50 of 50 nM. The chlorine substitution was well tolerated (compound2a, 220 nM), but was not as potent as 1i. Fluorine or methoxysubstitution at C-3 resulted in a loss of activity of more than 2 ordersof magnitude. C-4 substitution with bromine, chlorine, and methoxygroups all resulted in a decrease of activity of more than 2 orders ofmagnitude. Several disubstituted analogues were synthesized.3,4-Dichloro (2i) or 3,5-bis-(trifluoromethyl) (2h) substitutionresulted in potent inhibitors with IC50 values of 20 nM, about a 5-foldincrease in activity over 1i. The 3,5-dichloro substitution was alsotolerated. Dichlorine substituents at C-2 (2,3-Cl₂ or 2,4-Cl₂) resultedin poor activity, consistent with the aforementioned observation thatC-2 is a suboptimal site for substitution.

Varying Ethyl Groups at C-5 (FIG. 4).

The IC50 of the compounds decreased from 1300 nM for a hydrogen (3a) to200 nM for a methyl (3b) to 100 nM for an ethyl group (1i) at C-5. Thesame trend was observed for other substituents such as trifluoromethyl,bis(trifluoromethyl), and dichloro groups. Compared to those ofunsubstituted compounds and methyl-substituted compounds, the IC50 ofethyl-substituted compounds improved more than 10-fold and 2-3-fold,respectively. Changing the phenyl ring to bromo- or chloro-substitutedthiophenes resulted in a significant loss of activity of ˜20-fold.

Cyclized Pyrazoline Analogues (FIG. 5).

A set of analogues with the C-5 ethyl group attached to the C-3 nitrogento form a pyrazoline ring was synthesized to investigate the influenceof the restricted flexibility and the impact of substitution on the C-3nitrogen. Compound 4a resulted in a 2-fold decrease in activity ascompared to 1i. However, addition of a methyl group on the pyrazolinering (4b) restored the activity. Compound 4b had an IC50 of 80 nM,slightly better than that of the parent compound 1i. Similar trends wereobserved in the chlorine-substituted (4c, 4d) andtrifluoromethyl-substituted (4g, 4h) analogues. For dichlorosubstitutions, an additional methyl group in the ring (4f) reduced theactivity. Limits in solubility may contribute to the decreased activityof this compound. Compound 4f has the poorest solubility in methanolcompared to the others. Overall, there are five pyrazoline analogues(4b, 4e, 4f, 4g, and 4h) that have IC50 values better than that of 1i.

N1 NH2 Substitutions (FIG. 6).

A variety of noncyclized and cyclized pyrazoline thio semicarbazonessubstituted on the N1 amino group were synthesized. These included alkylgroups (e.g., methyl, dimethyl, ethyl, hexyl), aryl groups (e.g.,phenyl, chlorophenyl, trifluoromethylphenyl), methylfuran, andethylmorpholine. Most of the compounds had an IC50 greater than 10 μM. Afew of them (5c, 5d, 5e, and 5f) had moderate activity between 1 and 2μM.

C═S Double Bond (FIG. 7).

Semicarbazone analogues of the most active thio semicarbazones weresynthesized. All of these compounds had IC50 values greater than 10 μM.This is evidence that the sulfur in the C═S double bond is critical foractivity.

C═N4 Double Bond (Scheme 3).

Reduction of 1i by sodium borohydride generated the saturated C—N4 bondvariant of 1i (7a). Compound 7a exhibited poor activity compared to 1i,indicating the importance of the double bond.

Varying C5-groups with Longer Alkyl Chains and Aryl Groups (FIG. 14)

While the variation at C5 from H to CH₃ to CH₂CH₃ was obvious, longeralkyls such as propyl, butyl or other groups such as allyl, benzyl,phenylethyl group did not increase the IC₅₀ further, though generallyall the compounds have good activity. This is consistent with ourmodeling results. Since the S1 pocket is small, the maximum interactionis reached by having approximately two carbons. While additional carbonssimply project from the pocket. Depending on the sterics and rigidity ofthe group, the activity varies. Propyl and butyl provide a flexible fit,resulting in good activity.

Summary of Protease Inhibition Assays

The thio semicarbazone series exhibited a specific and consistentstructure-activity relationship. Reduction of the C═N4 bond or a changeof the C═S bond into a C═O bond results in poor activity. For compoundswith single phenyl rings, substitution at specific positions can lead toenhanced activity. The most potent compounds have at least onesubstituent group at C-3. Among the C-3 substituents, trifluoromethyl,bromine, and chlorine result in potent inhibitors while compounds withother groups have poor activity. Disubstitution with 3,4-dichlorine or3,5-bis(trifluoromethyl) results in potent inhibitors of cruzain. Bycontrast, C-2 or C-4 substitution alone is not useful. Five compounds(2b, 2h, 2i, 3f, 3h) have better IC50 values than 1i. Compounds with thesix phenyl-five heteroaromatic group also are effective inhibitors ofcruzain. Ethyl substitution at C-5 is favorable over a broad range ofcompounds with different aryl substituents. Connection of the C-5 ethylgroup and the N-3 into cyclized pyrazoline analogues resulted incompounds with potent activities. Some of the pyazoline compounds haveimproved IC50 values when compared with the noncyclized analogues.

Example 10 Inhibition of Rhodesain and Cathepsin B

The following example shows the activity of selected thio semi carbazonecompounds against rhodesain, the major protease of Trypanosoma bruceiand against human cathepsin B, a cancer and inflammatory disease target.

Enzyme assays to test the ability of thio semicarbazone compounds toinhibit the cysteine protease activity of rhodesain and cathepsin B werecarried out as described for assays to test inhibition of proteaseactivity by cruzain. In these assays, inhibition of cruzain is includedas a positive control. TABLE 1 Inhibition of Rhodesain and Cathepsin Bcathepsin B cathepsin B cruzain IC50 (μM) IC50 (μM) IC50 (μM) rhodesaincompound 5 min preincubation 30 min preincubation 5 min preincubationIC50 (μM) guo4-52 5 1 0.01 0.03 guo4-44 >>10 10 0.03 0.052du2-79 >>10 >>10 0.08 0.07 du5-13 >>10 10 0.1  0.06 du5-7 >>10 10 0.010.03 K002 0.5 <0.1 not doneInhibition to falcipain-2 and parasite P. falciparumCompound 2i: IC50 150 nM on falcipain-2.Compound 1i IC50 of parasite P. falciparum 16-32 μM

Example 11 Anti-Trypanosomal Assays in Cell Culture

Methods

Drug Screening in Cell Culture. Mammalian cells are routinely culturedin RPMI-1640 medium supplemented with 5-10% heat-inactivated fetal calfserum (FCS) at 37° C. in 5% CO₂. The Y strain of T. cruzi is maintainedby serial passage in bovine embryo skeletal muscle (BESM) cells.Infectious trypomastigotes are collected from culture supernatants. Fordrug assays, J774 macrophages were irradiated (5000 rad) and plated ontosix-well tissue culture plates 24 h prior to infection with ˜106trypomastigotes/well. Parasites are removed 2 h postinfection, and themedium is supplemented with the appropriate cysteine protease inhibitor(10 μM). Inhibitor stocks (10 mM) in DMSO were stored at 4° C. J774monolayers treated with a blank containing DMSO are used as a negativecontrol, and monolayers treated with a known trypanocidal inhibitor, 10μM N-methyl piperazine-Phe-homoPhe-vinyl sulfone phenyl(N-Pip-F-hF-VSPh) acted as a positive control (Engel et al., J. Exp.Med., 188:725-734 (1998)). RPMI medium with or without inhibitor isreplaced every 48 h. Cultures are maintained for up to 30 days andmonitored daily by contrast phase microscopy. T. cruzi completed theintracellular cycle in 5-6 days in the untreated controls but was unableto survive in macrophages treated with N-Pip-F-hF-VSPh. The comparativeeffectiveness of each inhibitor was estimated from plots of the durationof the intracellular cycle of T. cruzi (days) in treated vs untreatedcontrol wells.

Results

In addition to their effect on enzymatic activity, we determined whetherthio semithiocarbazones other than compound 1i entered cells and exerteda trypanocidal effect against this intracellular parasite.

Three (1i, 1e, 1f) of the most effective compounds were tested againstintact trypanosomes in cell culture. Typically, infected host cells diewithin 5 days without treatment. In contrast infected host cells treatedwith compound 1e (5 μM) survived 10 days, but the therapeutic indexassociated with this compound was low, with toxicity to host cellsobserved at 10 μM. Infected host cells treated with compound 1f survived14 days, but this compound crystallized in the cell culture medium. Inother words, compounds 1e and 1f were trypanostatic. Compound 1iexhibited significant trypanocidal activity without toxicity to hostcells or solubility problems. Infected cells treated with 1i at 10 μMwere cured of trypanosomal infection. To confirm trypanocidal activity,infected cells were treated with 1i for three weeks and then 1i wasremoved from the cell culture. No parasites were observed in either theculture supernatants or host cells. Cells were healthy and parasite-freeuntil the experiment was terminated at 6.5 weeks postinfection.

Encouraged by this result we explored compounds related to 1i in aneffort to find more active, trypanocidal analogues.

The trypanocidal properties of representative compounds as judged by thesurvival of infected host cells is shown in FIG. 8. The toxicity ofcompounds to mammalian host cells was also evaluated in the assay. Ofthe five 3-bromo-substituted compounds, three (1i, 3b, 4b) exhibited atrypanocidal effect. No parasites were observed in supernatants or hostcells after the inhibitors were removed at day 22. The3-chloro-substituted compound 2a was also trypanocidal, but the twodisubstituted compounds 2i and 2h were not, although one of them wastrypanostatic for 20 days. Three more compounds from the initial groupof thio semicarbazones were tested but were not effective. Toxicity (1c,1n) or solubility (1b) appears to be a problem for some of the compoundstested in this latter group. Two moderate inhibitors with substitutionon the N1 amino group (5c, 5d) had no effect on the parasites.

A second group of inhibitors having low IC50 values was evaluated bycell culture assay and these are shown in FIG. 9. Among these 10compounds, two (4c, 4g) were inactive. Three were trypanostatic (3f, 3d,4f). The remaining five compounds (2b, 3h, 4d, 4e, 4h) were trypanocidaland cure the infected cells. Altogether, nine compounds have beenidentified to be trypanocidal through the cell culture assay, indicatingthat the thio semicarbazone is a productive scaffold forantitrypanosomal therapy.

Summary of Cell Culture Anti-Trypanosomal Assays

Compounds that are potent cruzain inhibitors in vitro are notnecessarily active in cell culture. Antiparasitic inhibitors must beable to cross the macrophage's cell membrane and cross the parasite'scytoplasm in sufficient quantity to significantly inhibit cruzainwithout killing the host cell. Nine compounds in the thio semicarbazoneseries (three single-phenyl-ring-substituted compounds (1i, 2a, 2b), twoof the methyl-substituted thio semicarbazones (3b, 3h), and fourpyrazoline analogues (4b, 4d, 4e, 4h) are trypanocidal.

Lipinski described desired ranges for certain properties thought to beimportant for pharmacokinetics and drug development. They are C log P<5,number of hydrogen bond donors ≦5, number of hydrogen bond acceptors≦10, and molecular weight <500 (Lipinski, C. A. et al., Adv. DrugDelivery Rev., 23:3-25 (1997)). A compound that fulfills at least threeout of the four criteria adheres to Lipinski's rule. Table 1 lists suchproperties of the nine trypanocidal compounds. All of our most potentantiparasitic agents are fully compatible with Lipinski's rule. TABLE 1The Trypanocidal Compounds Have Physical Properties Compatible withReasonable Pharmacokinetics and Drug Availability no. of H no. of H no.of ID mol wt C log P bond acceptors bond acceptors Criteria met rule<500 <5 <5 <10 at least 3 1i 286 3.79 3 3 all 2a 242 3.64 3 3 all 2b 2753.81 3 3 all 3b 272 3.26 3 3 all 3h 262 3.71 3 3 all 4b 298 4.33 2 3 all4d 254 4.18 2 3 all 4e 274 4.25 2 3 all 4h 287 4.35 2 3 all*For Structures, See FIGS. 8 and 9

Example 12 Mechanism of Inhibition

The most active thio semicarbazone compounds including the pyrazolineanalogues are time-dependent inhibitors. This indicates that inhibitionby the thio semicarbazone series is mechanism-based. A variety ofcompounds including some of the ones with moderate to poor activity werechosen for a more detailed study of time-dependent inhibition (FIG. 10).A known irreversible inhibitor, K002 (morpholino urea-Phe-homoPhe-vinylsulfone benzene; Axys Pharmaceuticals Inc., South San Francisco,Calif.), and a known reversible covalent inhibitor, leupeptin, were usedas controls for the thio semicarbazones. Both controls were rapidlytime-dependent. Inhibitors with a free N1 amino group such as 1i, 4b,3a, 3b, and 4a are time-dependent. Even some of the weak inhibitors suchas 5e and 7a showed time dependency. The time dependency of 7a indicatesthat the C═N4 double bond is not the site for covalent bond formation.Thus, the only logical site for covalent interaction with cruzain in 7ais the C═S double bond.

Example 13 Molecular Modeling

Methods

DOCK 4.0.1 is used to position putative inhibitors in the enzyme activesite and score the quality of the interactions of inhibitors withcruzain (Ewing, T. J. A. et al., J. Comput. Chem., 18:1175-1189 (1997)).The procedure for constructing the molecular surface and the energy gridof the active site of cruzain is found in Du, et al. (Chem. Biol. (2000)7:733). The C log P values are calculated for each compound using thesoftware C Log P 4.61 (Daylight Chemical Information Systems, Santa Fe,N. Mex.) on the basis of the work of Hansch (Hansch. C. et al.,Substituent Constants for Correlation Analysis in Chemistry and Biology,Wiley-Interscience: New York, (1979)).

Results

To further understand the mechanism of interaction, computational toolswere used to dock compound 1i into the active site of cruzain. Thesemodels provide insight into the activity of the cruzain inhibitors. Thecalculated best orientation of 1i with cruzain is shown in FIG. 3. Thebromophenyl portion of 1i is oriented toward the deep S2 pocket. Such aninteraction is not ideal as judged by the modeling effort. We anticipatethat the ethyl group fits into the shallow S1 pocket, while the rest ofthe thio semicarbazone scaffold is positioned close to Cys25 and His159.The distance between one of the His159 protons and the sulfur in thethio semicarbazone is calculated to be 3.14 Å. In this dockedorientation, the distance between the Cys25 thiolate and the carbon (C2)attached to sulfur is 3.78 Å and the distance between the Cys25 thiolateand the carbon in C═N4 is 4.07 Å. The separation between N4 and theproton of His 159 (4.31 Å) is not compatible with a direct interaction.Therefore, this orientation of 1i suggests that the covalent attack ofthe Cys25 on 1i is directed toward the C2═S bond, consistent with thetime dependency of 7a. The attack on the C2═S bond would be assisted bythe transfer of the His159 proton to the thio semicarbazone sulfur (FIG.12).

If the ethyl group fits into the S1 pocket, this would explain thepreference for an ethyl group over a methyl group or a hydrogen in theS1 pocket. The unsubstituted pyrazoline ring analogues have a methylgroup equivalent on C-4. This observation is consistent with the resultthat the pyrazoline analogue 4a has 2-fold lower activity when comparedto the parent ethyl compound 1i. Upon addition of a methyl group to theC4 position on the pyrazoline ring, this analogue resembles the originalethyl substituent and its activity is restored.

When the semicarbazone analogue 6a was docked into the active site, noneof the top 20 orientations were similar to that of 1i. Most orient thesemicarbazone portion toward the S2 site and the aryl group toward theS′ site. In other words, the modeling suggests that the electronic andvolumetric difference between a sulfur and an oxygen atom may make asubstantial difference. This is consistent with the substantial activitydifference between compounds 6a and 1i.

Example 14 In Vitro Data for Compounds

Thio semicarbazones have been tested against falcipain 2, which is thecysteine protease of parasites related to malaria. Of those tested,compound 2i is the most promising, with an IC50 of 150 nM. The WHO(World Health Organization) helped to screen compound 1i against acouple of parasite strains. It has activity against T. cruzi and P.falciparum parasites. See FIG. 15.

1. A compound having the formula:

wherein R¹ is a member selected from the group consisting of substitutedor unsubstituted aryl and substituted or unsubstituted heteroaryl; R² issubstituted or unsubstituted lower alkyl; R³ is a member selected fromthe group consisting of H, and substituted or unsubstituted lower alkyl,and R² and R³ are optionally joined to form a ring system having theformula:

R⁴ is a member selected from H and substituted or unsubstituted loweralkyl; R⁵ is a member selected from the group consisting of H, andsubstituted or unsubstituted alkyl; and X is O or S.
 2. The compoundaccording to claim 1, wherein R¹ is a member selected from the groupconsisting of:

wherein R⁵, R⁶, and R⁷ are members independently selected from the groupconsisting of H, substituted or unsubstituted alkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, haloalkyl,alkoxy and halo; and Z is S or O.
 3. The compound according to claim 2,wherein R¹ is:

wherein R⁵, R⁶ and R⁷ are members independently selected from H, haloand haloalkyl.
 4. The compound according to claim 3, wherein R⁵ is H orhaloalkyl; R⁶ is H or halo; and R⁷ is halo or CF₃.
 5. The compoundaccording to claim 1, wherein R² is a member selected from the groupconsisting of H, CH₃, and CH₂CH₃.
 6. The compound according to claim 1,wherein R⁵ is a member selected from the group consisting of H, CH₃, andCH₂CH₃.
 7. A pharmaceutical composition comprising a therapeuticallyeffective amount of a compound of claim 1 and a physiologicallyacceptable carrier.
 8. The pharmaceutical composition of claim 7, saidcomposition comprising a therapeutically effective amount of a compoundselected from the group consisting of: a) 3′-Bromopropiophenone ThioSemicarbazone (1i), b) 3′-Chloropropiophenone Thio Semicarbazone (2a),c) 3′-Trifluoromethylpropiophenone Thio Semicarbazone (2b), d)3′-Bromoacetophenone Thio Semicarbazone (3b), e)3,4-Dichlorobenzaldehyde Thio Semicarbazone (3g) f)3′,4′-Dichloroacetophenone Thio Semicarbazone (3h), g)3-(3-Bromophenyl)-4-methyl-2-pyrazoline-1-thiocarboxamide (4b), h)3-(3-Chlorophenyl)-4-methyl-2-pyrazoline-1-thiocarboxamide (4d), i)3-(3,4-Dichlorophenyl)-2-pyrazoline-1-thiocarboxamide (4e), j)3-(3-Trifluoromethylphenyl)-4-methyl-2-pyrazoline-1-thiocarboxamide(4h), k) 3′,5′-bis(trifluoromethyl)propiophenone Thio Semicarbazone(2h), l) 3′,4′-Dichloropropiophenone Thio Semicarbazone (2i), m)3-Trifluoromethylacetophenone Thio Semicarbazone (3d), n)3′,5′-Bis(trifluoromethyl)acetophenone Thio Semicarbazone (3f), o)3-(3,4-Dichlorophenyl)-4-methyl-2-pyrazoline-1-thiocarboxamide (4f), andp) 3-(3-Trifluoromethylphenyl)-2-pyrazoline-1-thiocarboxamide (4g). 9.The pharmaceutical composition of claim 7, the composition comprising atherapeutically effective amount of a second compound.
 10. Thepharmaceutical composition of claim 7, wherein the composition isformulated for oral administration.
 11. The pharmaceutical compositionof claim 7, wherein the composition is formulated for parenteraladministration.
 12. A method of inhibiting a cysteine protease involvedin the infectious life cycle of a protozoa, said method comprising thesteps of: contacting said cysteine protease with a compound of claim 1,wherein said compound forms a reversible covalent interaction with acysteine in the active site of said cysteine protease, whereby saidcysteine protease is inhibited.
 13. The method of claim 12, wherein saidcompound is selected from the group consisting of: a)3′-Bromopropiophenone Thio Semicarbazone (1i), b) 3′-ChloropropiophenoneThio Semicarbazone (2a), c) 3′-Trifluoromethylpropiophenone ThioSemicarbazone (2b), d) 3′-Bromoacetophenone Thio Semicarbazone (3b), e)3,4-Dichlorobenzaldehyde Thio Semicarbazone (3g) f)3′,4′-Dichloroacetophenone Thio Semicarbazone (3h), g)3-(3-Bromophenyl)-4-methyl-2-pyrazoline-1-thiocarboxamide (4b), h)3-(3-Chlorophenyl)-4-methyl-2-pyrazoline-1-thiocarboxamide (4d), i)3-(3,4-Dichlorophenyl)-2-pyrazoline-1-thiocarboxamide (4e), and j)3-(3-Trifluoromethylphenyl)-4-methyl-2-pyrazoline-1-thiocarboxamide(4h), k) 3′,5′-bis(trifluoromethyl)propiophenone Thio Semicarbazone(2h), l) 3′,4′-Dichloropropiophenone Thio Semicarbazone (2i), m)3-Trifluoromethylacetophenone Thio Semicarbazone (3d), n)3′,5′-Bis(trifluoromethyl)acetophenone Thio Semicarbazone (3f), o)3-(3,4-Dichlorophenyl)-4-methyl-2-pyrazoline-1-thiocarboxamide (4f), p)3-(3-Trifluoromethylphenyl)-2-pyrazoline-1-thiocarboxamide (4g), q)3′-trifluoromethylbutyrophenone thio Semicarbazone (8f), r)3′-trifluoromethylvalerophenone thio Semicarbazone (8g), s)3-(3-trifluoromethylphenyl)-4-ethyl-2-pyrazoline-1-Thiocarboxamide (8k),and t)3-(3-trifluoromethylphenyl)-4-propyl-2-pyrazoline-1-Thiocarboxamide(8l).
 14. The method of claim 12, wherein said protozoan is aTrypanosoma, a Plasmodium or a Leishmania.
 15. A method of treating aparasitic disease, said method comprising the steps of: administering toa patient in need thereof a sufficient amount of a pharmaceuticalcomposition according to claim 7, wherein said compound inhibits acysteine protease involved in the infectious life cycle of a parasitecausing said parasitic disease, whereby said parasitic disease istreated.
 16. The method of claim 15, wherein said parasite is selectedfrom the group consisting of Trypanosoma, Plasmodium and Leishmania, andsaid parasitic disease is selected from the group consisting of Chagas'disease, African sleeping sickness, nagana, malaria, and leishmaniasis.17. The method of claim 15, wherein the patient is a human.
 18. A methodof preventing a parasitic infection, said method comprising the stepsof: administering to a patient in need thereof a sufficient amount of apharmaceutical composition according to claim 7, wherein said compoundinhibits a cysteine protease involved in the infectious life cycle of aparasite causing said parasitic disease, whereby said parasiticinfection is prevented.
 19. The method of claim 18, wherein saidparasite is selected from the group consisting of Trypanosoma,Plasmodium and Leishmania, and said parasitic disease is selected fromthe group consisting of Chagas' disease, African sleeping sickness,nagana, malaria, and leishmaniasis.
 20. The method of claim 18, whereinthe patient is a human.
 21. A method of inhibiting a mammalian cysteineprotease involved in the malignancy of a cancer cell, said methodcomprising the step of: contacting said cysteine protease with acompound of claim 1, wherein said compound forms a reversible covalentinteraction with a cysteine in the active site of said cysteineprotease, wherein said cysteine protease is inhibited.
 22. The methodaccording to claim 21, wherein said cysteine protease is selected fromthe group consisting of cathepsin B and cathepsin L.
 23. A method oftreating or preventing cancer, said method comprising the step of:administering to a patient in need thereof a sufficient amount of apharmaceutical composition comprising a compound of claim 1, whereinsaid compound forms a reversible covalent interaction with a cysteine inthe active site of said cysteine protease, wherein said cysteineprotease is inhibited.
 24. A method of inhibiting a cysteine proteaseinvolved in the infectious life cycle of a trypanosome, said methodcomprising the step of: contacting said cysteine protease with acompound selected from the group consisting of: a) 3′-BromopropiophenoneThio Semicarbazone (1i), b) 3′-Chloropropiophenone Thio Semicarbazone(2a), c) 3′-Trifluoromethylpropiophenone Thio Semicarbazone (2b), d)3′-Bromoacetophenone Thio Semicarbazone (3b), e)3,4-Dichlorobenzaldehyde Thio Semicarbazone (3g) f)3′,4′-Dichloroacetophenone Thio Semicarbazone (3h), g)3-(3-Bromophenyl)-4-methyl-2-pyrazoline-1-thiocarboxamide (4b), h)3-(3-Chlorophenyl)-4-methyl-2-pyrazoline-1-thiocarboxamide (4d), i)3-(3,4-Dichlorophenyl)-2-pyrazoline-1-thiocarboxamide (4e), and j)3-(3-Trifluoromethylphenyl)-4-methyl-2-pyrazoline-1-thiocarboxamide(4h), k) 3′,5′-bis(trifluoromethyl)propiophenone Thio Semicarbazone(2h), l) 3′,4′-Dichloropropiophenone Thio Semicarbazone (2i), m)3-Trifluoromethylacetophenone Thio Semicarbazone (3d), n)3′,5′-Bis(trifluoromethyl)acetophenone Thio Semicarbazone (3f), o)3-(3,4-Dichlorophenyl)-4-methyl-2-pyrazoline-1-thiocarboxamide (4f), andp) 3-(3-Trifluoromethylphenyl)-2-pyrazoline-1-thiocarboxamide (4g),wherein said compound forms a reversible covalent interaction with acysteine in the active site of said cysteine protease, whereby saidcysteine protease is inhibited.
 25. A method of treating a trypanosomalinfection, said method comprising the step of: administering to apatient in need thereof a sufficient amount of a pharmaceuticalcomposition comprising a compound selected from the group consisting of:a) 3′-Bromopropiophenone Thio Semicarbazone (1i), b)3′-Chloropropiophenone Thio Semicarbazone (2a), c)3′-Trifluoromethylpropiophenone Thio Semicarbazone (2b), d)3′-Bromoacetophenone Thio Semicarbazone (3b), e)3,4-Dichlorobenzaldehyde Thio Semicarbazone (3g) f)3′,4′-Dichloroacetophenone Thio Semicarbazone (3h), g)3-(3-Bromophenyl)-4-methyl-2-pyrazoline-1-thiocarboxamide (4b), h)3-(3-Chlorophenyl)-4-methyl-2-pyrazoline-1-thiocarboxamide (4d), i)3-(3,4-Dichlorophenyl)-2-pyrazoline-1-thiocarboxamide (4e), and j)3-(3-Trifluoromethylphenyl)-4-methyl-2-pyrazoline-1-thiocarboxamide(4h), k) 3′,5′-bis(trifluoromethyl)propiophenone Thio Semicarbazone(2h), l) 3′,4′-Dichloropropiophenone Thio Semicarbazone (2i), m)3-Trifluoromethylacetophenone Thio Semicarbazone (3d), n)3′,5′-Bis(trifluoromethyl)acetophenone Thio Semicarbazone (3f), o)3-(3,4-Dichlorophenyl)-4-methyl-2-pyrazoline-1-thiocarboxamide (4f), p)3-(3-Trifluoromethylphenyl)-2-pyrazoline-1-thiocarboxamide (4g), q)3′-trifluoromethylbutyrophenone thio Semicarbazone (8f), r)3′-trifluoromethylvalerophenone thio Semicarbazone (8g), s)3-(3-trifluoromethylphenyl)-4-ethyl-2-pyrazoline-1-Thiocarboxamide (8k),and t)3-(3-trifluoromethylphenyl)-4-propyl-2-pyrazoline-1-Thiocarboxamide(8l); wherein said compound inhibits a cysteine protease involved in theinfectious life cycle of a trypanosome causing said trypanosomalinfection, whereby said trypanosomal infection is treated.
 26. Themethod of claim 25, wherein the patient is a human.